PEPTIDES CAPABLE OF REACTIVATING p53 MUTANTS

ABSTRACT

The invention provides peptides that can reactivate p53 mutants efficiently and specifically, as well as methods that allow the identification, selection and isolation of such peptides, in a precise, cost and time effective manner. In particular, there are provided mutant p53 reactivating peptides that can restore the native wild type p53 folding, and hence the tumor suppressor activity, to the mutant p53 protein. Such peptides are useful for treating various conditions and diseases in which p53 is mutated.

RELATED APPLICATIONS

This application is a Continuation In Part of PCT Patent Application No.PCT/IB2014/063777 having International filing date of Aug. 7, 2014,which claims the benefit of priority under 35 USC §119(e) of U.S.Provisional Patent Application No. 61/862,977 filed on Aug. 7, 2013. Thecontents of the above applications are all incorporated by reference asif fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 65024SequenceListing.txt, created on Feb. 2,2016, comprising 80,538 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to peptides capable of reactivating mutant p53proteins, and use thereof in therapy.

BACKGROUND OF THE INVENTION

Cancer is a leading cause of death in developed countries, and as theaverage age of the population continues to rise, so do the numbers ofdiagnosed cases and economic implications. Cancer is not a singledisease, but rather a group of more than 200 diseases characterized byuncontrolled growth and spread of abnormal cells. Cancer is a highlyheterogeneous disease with major molecular differences in the expressionand distribution of tumor cell surface markers even among patients withthe same type and grade of cancer. Moreover, cellular mutations tend toaccumulate as cancer progresses, further increasing tumor heterogeneity.Most tumor cells exhibit genomic instability with an increasedexpression of oncogenes and inactivation of tumor suppressor genes.

The p53 gene is considered to be the most important tumor suppressorgene, which acts as a major barrier against cancer progression. The p53protein responds to various types of cellular stress, and triggers cellcycle arrest, apoptosis, or senescence (Levine, J. A., p53, the cellulargatekeeper for growth and division. Cell, 1997. 88: p. 323-331). This isachieved by transcriptional transactivation of specific target genescarrying p53 DNA binding motifs. It is widely agreed that the p53pathway is impaired in almost all human cancers. Mutation of p53 isviewed as a critical step in malignant transformation process and over50% of cancer cases carry mutations in their p53 genes. Most of thesemutations are missense point mutations that target the DNA-binding coredomain (DBD) of p53, thereby abolishing specific DNA binding of p53 toits target site. These mutations prevent p53-dependent transcription andconsequently p53-mediated tumor suppression. The exceptionally highfrequency of p53 mutations in human tumors of diverse types makes p53unique among genes involved in tumor development, rendering mutated p53(Mut-p53) an attractive target for novel cancer therapies.

Structural studies have revealed that the tumor-derived missensemutations in the DBD of p53 produce a common effect: destabilization ofDBD folding at physiological temperature (Joerger, A. C., M. D. Allen,and A. R. Fersht, Crystal structure of a superstable mutant of human p53core domain. Insights into the mechanism of rescuing oncogenicmutations. J Biol Chem, 2004. 279(2): p. 1291-6). This destabilizationmay be reversible, since some mutants can revert to wild-typeconformation and bind DNA at reduced temperatures. Thus, most mutationsof p53 destabilize p53 protein folding, causing partial denaturation atphysiological temperature.

Mutant p53 proteins accumulate at high levels in tumor cells, mainly dueto their inability to upregulate the expression of p53's own destructorMdm2. Moreover, many p53 activating stress signals (like hypoxia,genomic instability and oncogene expression) are constitutively inducedin cancer cells. Therefore, reactivation of Mut-p53 is expected to exertmajor anti-tumor effects. Furthermore, it has been shown in a mousemodel that restoration of p53 functions is well tolerated in normaltissues and produces no visible toxic effects (Ventura, A., et al.,Restoration of p53 function leads to tumour regression in vivo. Nature,2007. 445(7128): p. 661-5).

p53 has evolved to be dynamic and conformationally unstable. The lack ofa rigid structure of the p53 protein may result in a number of p53conformers displaying different activity, depending on the type ofstress and cellular context. In a simplified model, p53 can assumeeither a wild type, active conformation or a mutant, misfolded, inactiveconformation. The two conformational states of p53 can be distinguishedby two specific monoclonal antibodies, PAb240 and PAb1620 (Wang, P. L.,F. Sait, and G. Winter, The ‘wild type’ conformation of p53: epitopemapping using hybrid proteins. Oncogene, 2001. 20(18): p. 2318-24).PAb240 binds to residues 212-217 in the DBD of p53. This region isinaccessible to the antibody (Ab) in the wild type (WT) conformation.However, in denatured or mutant p53, it is exposed (Vojtesek, B., etal., Conformational changes in p53 analyzed using new antibodies to thecore DNA binding domain of the protein. Oncogene, 1995. 10(2): p.389-93). PAb1620 recognizes a conformational, nonlinear epitope in theDBD, composed of two distinct regions of p53 and including residuesR156, L206, R209 and N210 (Cook, A. and J. Milner, Evidence forallosteric variants of wild-type p53, a tumor suppressor protein. Br JCancer, 1990. 61(4): p. 548-52). In the WT conformation the protein isfolded in a way that holds the loops in close proximity to each other(Ravera, M. W., et al., Identification of an allosteric binding site onthe transcription factor p53 using a phage-displayed peptide library.Oncogene, 1998. 16(15): p. 1993-9), forming the complete epitoperecognized by PAb1620. When p53 protein is misfolded (as a result ofmutation, temperature, denaturation or the like), these two loops movefarther away, the epitope is destroyed and therefore the mutantconformation is PAb1620 negative. It has been shown that p53 is aconformationally flexible protein. However, the defect in folding insuch mutants is not irreversible: some p53 mutants maintain residualDNA-binding ability, mutants that fail to bind DNA at 37° C. can bind atsub-physiological temperatures (32° C. or 25° C.), and activatetranscription from a p53-responsive promoter at 26° C. In addition, theisolated DBD's of mutant proteins R245S, R282W, V143A and others wereshown to have residual (30-60%) DNA-binding activity at 20° C.

Structural studies show that the extent of misfolding differs amongmutants; however, there is no defined alternative fold but rather apartial denaturation. This suggests that a “small molecule’ approach toreverse the effect of p53 mutation on folding could be applicable to awide range of mutant forms. Another important prediction from structuralstudies is that a ligand that binds to the properly folded fraction ofthe protein is expected to shift the equilibrium towards the native foldaccording to the law of mass action.

p53 was first identified as a cellular protein interacting with the SV40large T antigen (LT). The interface area between LT and p53 is large: atotal of 23 LT residues and 19 p53 residues are either buried in thisinterface or are found to directly participate in the interactionsbetween these two molecules. p53/DNA interaction residues are adjacentand overlapping with the p53/LT interface. The binding of LT to thesep53 residues can effectively shield the entire DNA-binding surface ofp53, including the three most commonly mutated p53 residues in cancer:R273, R248, and G245. This inhibits transactivation of p53-dependentpromoters. Since the p53/LT interface involves several different p53regions and loops, the p53 protein has to be folded correctly to alignamino acids in the correct location and orientation to form the bindingcontext to LT. Therefore, p53 binding to LT can serve as a marker to thep53 conformational state.

Several correctional approaches were attempted in the p53 conformationfield. Proof of principle for conformation stabilizing peptides wasprovided by Friedler and colleagues (Friedler, A., et al., A peptidethat binds and stabilizes p53 core domain: chaperone strategy for rescueof oncogenic mutants. Proc. Natl. Acad. Sci. USA, 2002. 99(2): p.937-42). A nine-residue peptide, CDB3, was designed based on the crystalstructure of the complex between the p53 DBD and ASPP (Samuels-Lev, Y.,et al., ASPP proteins specifically stimulate the apoptotic function ofp53. Mol. Cell, 2001. 8(4): p. 781-94). This peptide was shown to bindMut-p53 and act as a chaperone, shifting equilibrium towards the WTconformation, as indicated by increased reactivity to PAb1620. However,the biological effects of CDB3 (Issaeva, N., et al., Rescue of mutantsof the tumor suppressor p53 in cancer cells by a designed peptide. Proc.Natl. Acad. Sci. USA, 2003. 100(23): p. 13303-7) are only partial sincethe conformation of the Mut-p53/CDB3 complex is in an intermediate statebetween WT and mutant.

Small molecule compounds targeting Mut-p53 have been identified usingeither protein-based or cell-based assays (Peng, Y., et al., Rescue ofmutant p53 transcription function by ellipticine. Oncogene, 2003.22(29): p. 4478-87). CP-31398 was identified by screening for moleculesthat protect the isolated p53 DBD from thermal denaturation, as assessedby maintenance of PAb1620 reactivity upon protein heating (Foster, B.A., et al., Pharmacological rescue of mutant p53 conformation andfunction. Science, 1999. 286(5449): p. 2507-10). The mechanism of actionof CP-31398 remains unclear. NMR studies failed to detect any binding ofCP-31398 to the p53 DBD (Rippin, T. M., et al., Characterization of thep53-rescue drug CP-31398 in vitro and in living cells. Oncogene, 2002.21(14): p. 2119-29). CP-31398 affects gene expression and induces celldeath both in a p53-dependent and independent manner. Thus, it appearsthat CP-3138 has other cellular targets than p53 that may account forits cellular toxicity.

Two other small molecules that rescue p53 function in living cancercells, PRIMA-1 and MIRA-1, were discovered by using cell-based screeningassays. PRIMA-1 and MIRA-1 have similar activity profiles (Bykov, V. J.,et al., Reactivation of mutant p53 and induction of apoptosis in humantumor cells by maleimide analogs. J Biol Chem, 2005. 280(34): p.30384-91), but are structurally unrelated. So far, direct binding toMut-p53 has not been demonstrated. It seems that the mechanism mayinvolve the JNK pathway.

In the field of anti-cancer drug discovery and design, two different andat times complementary, strategies may be employed. Rational design,which uses biological, mathematical or computational tools to designmolecules for a certain purpose, has been used in the case of CDB3.However, since the interactions between different proteins and theirenvironment are complex, this is extremely difficult and often yieldsmolecules with a modest biological impact. The second strategy is highthroughput screening of molecule libraries, to isolate compounds withthe best traits. Such screening can employ either chemical, smallmolecule libraries or peptide libraries. Most drugs available to dateare small molecules because of their ability to cross cell membranes.Chemical libraries usually consistent of 10⁴-10⁵ different compounds;screening such a library requires functional assessment of individualmolecules, making it impractical for a small laboratory since it callsfor large investments in robotics and/or manpower. Peptide displaylibraries are much larger. Selection of peptides is based on binding ofpeptides (and hence the phage), to an immobilized target, elution andamplification and then identification by sequencing.

In the phage display procedure, enrichment of phages that present apeptide is achieved by affinity selection of a phage library onimmobilized target. In this “panning” process, binding phages arecaptured whereas nonbinding ones are washed off. In the next step, thebound phages are eluted and amplified by reinfection of E. coli cells.The amplified phage population can, in turn, be subjected to the nextround of panning. The selection from phage display libraries is a cyclicprocess of selective enrichment and amplification. After several roundsof selection, phages are diluted in a way that allows isolation ofindividual phage clones. Individual clones are then picked, cultivatedin E-coli, phage DNA is extracted and then sent to sequencing. Recentlydeveloped next-generation sequencing technologies are greatly increasingthe effectiveness of phage display, allowing analysis of the entireselected peptide repertoire, with fewer selection rounds performed.

Phage display offers several important advantages over other screeningmethods; the major advantage of phage display is the diversity ofsequences that can be represented, enabling finding molecules with veryhigh affinity and biological effect. Once a consensus peptide sequenceis found, it can be further improved by either directed evolutiontechniques or rational design.

Nevertheless, there remains an unmet need in the art for agents that canreactivate p53 mutant proteins efficiently and specifically. Suchspecific and efficient agents can further be used as an effective meanfor treating various conditions in which p53 is mutated, in particular,by restoring the native folding and activity of the mutant p53 proteins.

SUMMARY OF THE INVENTION

The present invention provides highly potent peptides and modifiedpeptide agents that can efficiently reactivate p53 conformationalmutants, ideally by changing the mutant p53 proteins conformation and/oractivity to resemble that of a wild type, functional p53 protein. Thepresent invention thus provides peptides and their use in treatingmutant p53 related conditions, where activation of present yetconformationally defective p53 proteins may be beneficial.

The present invention is based on the surprising identification ofhighly potent peptide and peptide-based agents that can efficientlyreactivate p53 conformational mutants, more efficiently than previouslyknown peptides identified for this use. The present invention thusprovides, in an aspect, a recombinant or synthetic peptide consisting ofthe amino-acid sequence set forth in any one of SEQ ID NOs:321-286.

The present invention further provides, in another aspect, a recombinantor synthetic peptide comprising the amino-acid sequence set forth in anyone of SEQ ID NOs:321-286, wherein said peptide at least partiallyreactivates a mutant p53 protein.

The present invention further provides, in yet another aspect, arecombinant or synthetic peptide comprising the amino-acid sequence setforth in any one of SEQ ID NOs: 302-321, 312-321 and 316-321, whereinsaid peptide at least partially reactivates a mutant p53 protein. Eachpossibility represents a separate embodiment of the invention.

The present invention further provides, in yet another aspect, arecombinant or synthetic peptide comprising the amino-acid sequence setforth in any one of SEQ ID NOs: 316-321, wherein said peptide at leastpartially reactivates a mutant p53 protein. Each possibility representsa separate embodiment of the invention.

The present invention further provides, in yet another aspect, arecombinant or synthetic peptide comprising a consensus motif of theamino-acid sequence set forth in any one of SEQ ID NOs: 314, 268, 282,340, 376, 298, 377, 378, 253, 20, 379, 302, 275, 380, 273, 381, 280 or382, wherein said peptide at least partially reactivates a mutant p53protein. According to a specific embodiment the consensus motif is asset forth in SEQ ID NO: 314.

In certain embodiments, the peptide consists of the amino-acid sequenceset forth in any one of SEQ ID NO:321, SEQ ID NO:314, SEQ ID NO:313, SEQID NO:310 or SEQ ID NO:307. Each possibility represents a separateembodiment of the invention. In certain embodiments, the peptidedescribed above consists the amino-acid sequence set forth in any one ofSEQ ID NOs:321-302. Each possibility represents a separate embodiment ofthe invention. In certain embodiments, the peptide described aboveconsists the amino-acid sequence set forth in any one of SEQ IDNOs:321-312. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the peptide described above consiststhe amino-acid sequence set forth in any one of SEQ ID NOs:321-316. Eachpossibility represents a separate embodiment of the invention. Incertain embodiments, the peptide described above consists the amino-acidsequence set forth in any one of SEQ ID NOs: 302-321, 312-321 and316-321, wherein said peptide at least partially reactivates a mutantp53 protein. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the peptide described above consists theamino-acid sequence set forth in any one of SEQ ID NOs: 302-321, 312-321and 316-321, wherein said peptide at least partially reactivates amutant p53 protein. Each possibility represents a separate embodiment ofthe invention.

In certain embodiments, the peptide comprises of the amino-acid sequenceset forth in any one of SEQ ID NO:321, SEQ ID NO:314, SEQ ID NO:313, SEQID NO:310 or SEQ ID NO:307. Each possibility represents a separateembodiment of the invention. In certain embodiments, the peptidedescribed above comprises the amino-acid sequence set forth in any oneof SEQ ID NOs:321-302. In certain embodiments, the peptide describedabove comprises the amino-acid sequence set forth in any one of SEQ IDNOs:321-312. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the peptide described above comprisesthe amino-acid sequence set forth in any one of SEQ ID NOs:321-316. Eachpossibility represents a separate embodiment of the invention. Incertain embodiments, the peptide described above comprises theamino-acid sequence set forth in any one of SEQ ID NOs: 307, 310, 313,314 and 321. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the peptide described above comprises theamino-acid sequence set forth in any one of SEQ ID NOs: 302-321, 312-321and 316-321. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the peptide is conjugated to at least one cellpenetrating moiety (lipid and/or proteinaceous). The cell penetratingmoiety may be conjugated N-terminally to the peptide, C terminally tothe peptide, anywhere in the backbone of the peptide or to a combinationof same. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the cell penetrating moiety is selected from thegroup consisting of a fatty acid moiety, a protennacious moiety and acombination of same.

In certain embodiments, the peptide is conjugated to at least one fattyacid moiety. In certain embodiments, the fatty acid is selected from thegroup consisting of myristic acid, lauric acid, palmitic acid andstearic acid. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the fatty acid is a myristoyl fattyacid.

In certain embodiments, the peptide is conjugated to at least oneproteinacious moiety. In certain embodiments, the proteinaceous moietyis a poly-cationic amino acid e.g., poly-Lysine and/or poly-Argininee.g., having 2-15 arginine residues e.g., conjugated to at least one endof the peptide (N and/or C). Each possibility represents a separateembodiment of the invention. According to a specific embodiment, theprotenaceious moiety comprises at least one positively charged aminoacid at either of the peptide's termini i.e., N and/or C terminus. Eachpossibility represents a separate embodiment of the invention. Forinstance, at least one positively charged (e.g., R, RR, RRR) can beconjugated to the N-terminus or C-terminus of SEQ ID NO: 314. Eachpossibility represents a separate embodiment of the invention.

In certain embodiments, the peptide at least partially changes theconformation of said mutant p53 protein to a conformation of a wild-type(WT) p53 protein.

In certain embodiments, the peptide at least partially changes theconformation of said mutant p53 protein such that said mutant p53protein is recognized by a monoclonal antibody directed against a WT p53protein. In certain embodiments, the monoclonal antibody is Ab1620.

In certain embodiments, the peptide at least partially restores theactivity of said mutant p53 protein to the activity of a WT p53 protein.

In certain embodiments, the activity is reducing viability of cellsexpressing said mutant p53 protein. In certain embodiments, the activityis promoting apoptosis of cells expressing said mutant p53 protein. Incertain embodiments, the activity is activating pro-apoptotic genes ofcells expressing said mutant p53 protein. In certain embodiments, thepro-apoptotic genes are selected from the group consisting of CD95, Bax,DR4, DR5, PUMA, NOXA, Bid, 53AIP1 and PERP. Each possibility representsa separate embodiment of the invention.

In certain embodiments, the activity is binding to a p53 consensus DNAbinding element in cells expressing said mutant p53 protein. In certainembodiments, the consensus DNA binding element comprises the amino acidsequence set forth in SEQ ID NO:339.

In certain embodiments, the binding results in at least partialactivation of an endogenous p53 target gene. In certain embodiments, theendogenous target gene is selected from the group consisting of p21,MDM2 and PUMA. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the mutant p53 protein is of a differentconformation than a WT p53 protein. In certain embodiments, the mutantp53 protein is at least partly inactive compared to a WT p53 protein.

In certain embodiments, the mutant p53 protein is not recognized by amonoclonal antibody directed against a WT p53 protein. In certainembodiments, the mutant p53 protein, upon binding to said peptide, isrecognized by a monoclonal antibody directed against a WT p53 protein.In certain embodiments, the monoclonal antibody is Ab1620.

In certain embodiments, the mutant p53 protein comprises a mutationselected from the group consisting of R175H, V143A, R249S, R273H, R280K,P309S, P151S, P151H, C176S, C176F, H179L, Q192R, R213Q, Y220C, Y220D,R245S, R282W, D281G, S241F, C242R, R248Q, R248W, D281G, R273C and V274F.Each possibility represents a separate embodiment of the invention.

In certain embodiments, the peptide comprises the consensus motif setforth in SEQ ID NO:314. In certain embodiments, the peptide comprisesthe amino-acid sequence set forth in any one of SEQ ID NO:321, SEQ IDNO:314, SEQ ID NO:313, SEQ ID NO:310 or SEQ ID NO:307. Each possibilityrepresents a separate embodiment of the invention. In certainembodiments, the peptide consists of the amino-acid sequence set forthin any one of SEQ ID NO:321, SEQ ID NO:314, SEQ ID NO:313, SEQ ID NO:310or SEQ ID NO:307. Each possibility represents a separate embodiment ofthe invention. In certain embodiments, the peptide comprises theamino-acid sequence set forth in any one of SEQ ID NOs:268, 282, 340,376, 298, 377, 378, 253, 20, 379, 302, 275, 380, 273, 381, 280 or 382.Each possibility represents a separate embodiment of the invention. Incertain embodiments, the peptide comprises the amino-acid sequence setforth in any one of SEQ ID NOs:379, 302, 275, 380, 273, 381, 280 or 382.Each possibility represents a separate embodiment of the invention. Incertain embodiments, the peptide comprises the amino-acid sequence setforth in any one of SEQ ID NOs: SEQ ID NOs:302, 275, 380, 273, 381, 280or 382. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the peptide described above comprisesthe amino-acid sequence set forth in any one of SEQ ID NOs: 307, 310,313, 314 and 321. Each possibility represents a separate embodiment ofthe invention. In certain embodiments, the peptide described abovecomprises the amino-acid sequence set forth in any one of SEQ ID NOs:302-321, 312-321 and 316-321. Each possibility represents a separateembodiment of the invention.

The present invention further provides, in another aspect, an expressionvector, capable of expressing the peptides described above.

The present invention further provides, in yet another aspect, apharmaceutical composition, comprising the peptides described above.

The present invention further provides, in yet another aspect, apharmaceutical composition, comprising the expression vector describedabove.

In an aspect, the pharmaceutical compositions described above are foruse in treating a disease, disorder or condition associated with amutant p53 protein.

In some embodiments, the disease is cancer. In some embodiments, thecancer is selected from the group consisting of breast cancer, coloncancer and lung cancer. Each possibility represents a separateembodiment of the invention.

In some embodiments, the cells of the cancer express the mutant p53protein.

The present invention further provides, in another aspect, a method oftreating a disease, disorder or condition associated with a mutant p53protein, comprising the step of administering a therapeuticallyeffective amount of the pharmaceutical compositions described above to asubject in need thereof, thereby treating said disease, disorder orcondition.

The present invention further provides, in yet another aspect, a kitcomprising the pharmaceutical compositions described above.

In an aspect, the kit described above is for use in treating a disease,disorder or condition associated with a mutant p53 protein.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a block diagram of steps in a screening method, whichprovides for selection of binding partners (such as peptides), in anon-direct way, through their effect on conformation or structure of atarget molecule.

FIG. 1B is a schematic drawing of a method of identification, screeningand selection of mutant p53 reactivating peptides. The method comprisesalternating various selection strategies, at increasing stringencies, toscreen and identify mutant p53 reactivating peptides, by utilizing aphage display method. Strategy A (left): selection according toconformation: selection of peptides expressed and presented by a phage,which can bind a mutant p53 protein (for example, R175H Mut-p53). TheMut-p53 protein is bound to a specific p53 antibody (for example,PAb1620) that is immobilized to a substrate, thereby enabling selectionof a bound phage. Strategy B (right): selection according to function:selection of peptides expressed and presented by a phage, which canreactivate a Mut-p53 (for example, R175H Mut-p53), whereby theactivation is determined by the ability of the Mut-p53 protein to bindits DNA consensus binding element. The DNA binding element (for example,WT p53-RE) is immobilized to a substrate. A Mut-p53 cannot bind the WTp53-RE, unless it is at least partially reactivated by the reactivatingpeptide bound thereto. The method may further comprise sequencing (forexample, deep sequencing) of the identified peptides to determine theirsequences, and optionally identify a consensus sequence for reactivatingpeptides.

FIG. 2 is a pictogram of a western blot analysis of immunoprecipitation(IP) experiments, in which agarose beads covalently cross-linked toantibodies (PAb1620 or PAb240) or proteins (ASPP2 or Bcl2) were incubatewith a WT p53 protein, mutant p53 R175H protein or mutant p53 V143A(each produced from sf9 cells transfected with baculovirus expressingthe respective protein) for 3 hours at 4° C. The resultantimmunoprecipitate, as well as the supernatant (sup) were subjected towestern blot experiments, using an anti p53 (αp53) antibody conjugatedto horseradish peroxidase (HRP), to determine the p53 protein level ineach sample.

FIG. 3 is a pictograms of western blot analysis of IP experiments, inwhich beads that were covalently cross linked to PAb1620 or PAb240antibodies were incubated with WT p53 or mutant p53 R175H for 3 hours at4° C. with various solutions (A-I and IP buffer). The resultantimmunoprecipitate, as well as the supernatant (sup) were subjected towestern blot experiments, using an anti p53 (αp53) antibody conjugatedto HRP, to determine the p53 protein level in each sample. Solution A—50mM Tris; solution B—Tris, 150 mM NaCl; solution C—Tris, NaCl, 0.5%Triton; solution D—Tris, 0.5% Glycine; solution E—40 mM Na₄O₇P₂;solution F—400 mM Guanidine-HCl; solution G—800 mM Guanidine-HCl;solution H—1M Urea; solution I—3M Urea; IP—IP Buffer.

FIG. 4 is the sequence of the oligonucleotide used as the bindingelement for p53 proteins. The oligonucleotide (SEQ ID NO:61) comprises a5′ biotin label, followed by a HindIll recognition site (underlined),followed by EcoRI recognition site (underlined), followed by a p53consensus binding element (underlined, p53 binding site is composed oftwo half sites, each half site binds a dimmer of p53 and together thissite forms a complex of DNA and p53 tetramer), followed by two copies ofthe p53 recognition element of the p21 promoter (underlined). Forbinding experiments, this oligonucleotide was annealed to acomplementary oligonucleotide to form a double stranded (ds)oligonucleotide.

FIG. 5 is a pictograms of western blot analysis of IP experiments, inwhich beads that were covalently cross-linked to a PAb1620 antibody wereincubated with purified mutant p53 R175H in the presence of phageobtained by phage display selection with either full length Mut-p53R175H (175) or recombinant Mut-p53 R249S (249 DBD), with priorpre-clearing step performed by incubation of the phage pool with PAb1620beads. Non selected phage (NS) were used as control. Incubation was donefor 3 hours at 4° C. Bound p53 in the immunoprecipitate was analyzed bywestern blot analysis using antibody against p53 (αp53). Non selectedphage (NS) were used as control. “In” stands for 10% of the IP inputmaterial that was loaded directly on the gel Immunoprecipitation withthe PAb-421 was used as a positive control and as standard forimmunoprecipitated p53, since this antibody binds p53 epitope at theC-terminus regardless of p53 protein conformation.

FIG. 6 is a pictograms of western blot analysis of IP experiments, inwhich streptavidin-coated beads bound either to p53-RE-DNA orcontrol-RE-DNA oligonucleotides labeled with biotin were incubated withpurified WT-p53-DBD or mutant p53-R249S-DBD in the presence of phageobtained by phage display selection with Mut-p53 R175H (175), clone 27(a single clone isolated from the 175 selection, SEQ ID NO:328); pools#69 and #94 selected with WT and Mut-p53 R175H using combinations ofSV-40 large T antigen (T-ag) and PAb1620 at alternating selectionrounds. Non selected phage (NS) were used as control. Incubation wasperformed for 3 hours at 4° C. Bound p53 was visualized by western blotanalysis using antibody against p53 (αp53).

FIG. 7 is a schematic illustration of several consensus peptide motifsidentified as described herein.

FIGS. 8A and 8B are bar graphs, demonstrating representative ELISAexperiments of determining the effect of tested peptides on theconformation changes of Mut-p53 in H1299 cells stably overexpressingMut-p53 (R175H p53), as determined by immunoassay. To measure theconformational effect of the peptides on Mut-p53, a micro-titer platewas coated with either PAb240, PAb1620 or pAb421 (as a positive controland standard for total p53 protein, since the antibody used recognizesboth WT and mutant conformations), overnight incubated, washed, blocked,and cell extracts (with or without peptides) were added for anadditional 2 hours. After removal of extracts, plates were washed andincubated with the αp53-HRP conjugated Ab for the detection of p53levels. A TMB (substrate of HRP) assay was performed and optical densitywas determined at 450 nm WT p53 served as a positive control forreactivity with PAb1620, and Mut-p53 served as a negative control. Theresults are presented as the ratio of absorbance between the PAb1620 orPAb240 samples and the control pAb241 sample. MCF7 and H1299-Mut-p53(ts) A135V (TS) cells were used as positive controls for the WT p53conformation (1620/240 ratio equals or exceeds 5:1).

FIG. 9 is a bar graph, demonstrating representative ELISA experiments ofdetermining the effect of tested peptides on the DNA binding activity ofMut-p53 in H1299 cells stably overexpressing Mut-p53 (R175H p53). Acommercial p53/DNA binding kit (R&D) was used, according to manufacturerinstructions. Briefly, 96 well plates were coated with anti-p53 antibodyovernight. Cell extracts containing p53 were reacted with anoligonucleotide that contains a p53 consensus binding site, labeled withbiotin, in the presence or absence (NT) of test peptides. WT p53 isexpected to bind this DNA binding site as well as to the antibodycoating the test wells of the plate. Excess p53 and oligos are washedaway and streptavidin-HRP is used to quantify the amount of oligos inthe well, which is proportional to the DNA bound by p53. TMB assay wasperformed to determine HRP levels (450 nm). The results are presented asrelative absorbance (at 450 nm) (Y-Axis) of each tested sample. MCF7 andthe H1299-Mut-p53 (ts) A135V cells serve as positive controls for WTp53.

FIG. 10 is a bar graph depicting representative ELISA experiments todetermine the binding of tested peptides to recombinant WT p53 andMut-p53. A commercial peptide-protein binding kit (TAKARA) was usedaccording to the manufacturer's instructions. Briefly, 96 well plateswere coated with peptides for 2 hours. Soluble peptides were added tothe corresponding wells to serve as a competition control to confirm thespecificity of peptide binding to p53 (+comp). p53-RE DNA oligo wasadded to other wells (+DNA) to examine whether it affects the binding ofpeptides to p53. After removal of recombinant protein, plates werewashed and incubated with αp53-HRP conjugated Ab for quantification ofp53. Finally a TMB (substrate of HRP) assay was performed and opticaldensity was determined at 450 nm. The results are presented as relativeabsorbance at 450 nm (Y-Axis) of each tested sample. The following αp53monoclonal antibodies served as internal controls: PAb1801; PAb1620 andPAb240.

FIG. 11 is a bar graph, demonstrating binding of Mut-p53 to promoters ofrepresentative p53 target genes in live cells. BT-549 breast cancercells endogenously expressing mutant p53^(R249S) were treated for 5 hourwith a mix of 3 pCAPs—250, 308 and 325. Cells treated with a mix ofcontrol peptides (inert peptides) served as a negative control. Cellswere fixed with 1% formaldehyde, harvested and DNA was sheared bysonication. DNA cross-linked to p53 was immunoprecipitated using apolyclonal anti-p53 antibody (H47). DNA was purified and binding to thep53 responsive elements of the PUMA, p21, CD95 and MDM2 gene promoterswas quantified by qPCR. Results were normalized to input samples thatrepresent total DNA levels. As a negative control, extracts wereimmunoprecipitated with beads without antibody (beads). A genomic sitenot containing any p53 binding element served as a negative control(black).

FIG. 12 is a bar graph illustrating the relative luciferase activity(cLuc/gLuc) as measured in the various tested samples. Transienttransfection of H1299 p53^(−/−) cells was performed with plasmidsexpressing WT p53, R175H p53, R249S p53 or empty vector as control,together with TK-RGC-luc, where luciferase expression is under controlof a tandem array of multiple p53-responsive elements. 24 hours aftertransfection, cells were treated with the test peptides. 48 hours aftertransfection, a sample of the culture medium was taken forbioluminescence measurements.

FIGS. 13A and 13B are bar graphs illustrating the effect of varioustested peptides on the viability of cells expressing Mut-p53, asdetermine by crystal violet assay. WI-38 fibroblasts expressingendogenous WT p53 were infected with retroviruses expressing eithermouse Noxa shRNA (WI38-m-Noxa-i) as a nonspecific control or the R175Hp53 mutant for stable overexpression of mutant p53 (WI38-175). The cells(WI38-m-Noxa-i or WI38-175) were seeded at 3000 cells per well in96-well plates. Tested peptides were added to the cells. Differentconcentrations of etoposide (cytotoxic drug,4′-Demethyl-epipodophyllotoxin9-[4,6-O—(R)-ethylidene-beta-D-glucopyranoside], 4′-(dihydrogenphosphate) were used as positive control for cell death and as astandard reference curve to assess the effect of tested peptides. 48hours after treatment, cells were washed with PBS to exclude dead cellsand debris, and cells that remained attached to the plate were stainedwith crystal-violet for 30 minutes. Crystal violet was removed and cellswere washed 4 times with PBS to remove residual crystal violet. Then,the stained cells were dissolved in 10% acetic acid and plates weretaken for optical density measurement at 595 nM (optimal for crystalviolet). The bar graphs of FIGS. 13A and 13B show the optical densityreads at 595 nm, which reflect the number of cells in the plate aftertreatment, normalized to the non-treated (NT) samples.

FIG. 14 is a bar graph illustrating the effect of tested peptides onactivation of Mut-p53 by measuring transactivation of p53 target genesas determined by qRT-PCR. H1299 cells are p53 null and are widely usedfor p53 research. H1299 cells stably transfected with Mut-p53 (ts) A135Vwere used. The cells were plated in 12-well dishes, the indicatedpeptides were added directly to the medium at a concentration of 5ug/ml, and cells were then either moved to 32° C. or returned to 37° C.18 hours later cells were harvested, followed by extraction of RNA, cDNAsynthesis and real time PCR analysis. The expression level of 3representative p53 target genes, p21, PUMA and Mdm2, was examined. Thebar graphs shown in FIG. 14 illustrate the relative fold induction oftranscription of the tested genes in the various samples relative totheir transcription level in non-treated cells.

FIGS. 15A and 15B are bar graphs illustrating the effect of the variousindicated peptides on the viability of breast cancer cells expressingdifferent Mut-p53 isoforms, as determined by crystal violet assay. FIG.15A: MDA-MB-231 cells expressing Mut-p53 with a mutation at position 280of the DBD. FIG. 15B: SKBR3 cells expressing Mut-p53 with mutation atposition 175 within the DBD. The bar graphs in FIGS. 15A and 15B showfor each tested peptide the optical density reads at 595 nm, reflectingthe number of cells in the plate after treatment, normalized to thenon-treated (NT) samples.

FIG. 16 is a bar graph illustrating the effect of the indicated peptideson activation of Mut-p53 by measuring transactivation of p53 targetgenes as determined by qRT-PCR. SKBR3 ShCon cells and SKBR3 Shp53 cellsknocked down for p53 expression were used. The cells were plated in12-well dishes and the indicated peptides were added directly to themedium at a concentration of 5 ug/ml. 18 hours later cells wereharvested, followed by qRT-PCR analysis. Expression level of p21, PUMAand Mdm2 was evaluated. FIG. 16 illustrates the relative fold inductionof transcription of the tested genes in the various samples relative totheir transcription level in non-treated cells. GAPDH mRNA was measuredin parallel as a control.

FIGS. 17A, 17B, 17C and 17D illustrate representative experimentsperformed on ES2 ovarian carcinoma cells expressing Mut-p53 mutated atposition 241 within the DBD. In essence, the cells were plated in 6 cmdishes, and the indicated peptides were added directly to the medium ata concentration of 12 ug/ml at the indicated time points. Cells wereharvested and an apoptosis assay (FIGS. 17A and 17B) was performed usingthe Annexin-V staining kit (Roche, REF 11 988 549 001). Non-fixed cellswere stained with both anti Annexin FITC conjugated antibody to detectapoptotic cells, and PI (propidium iodide) to stain dead cells,according to the manufacturer's instructions. Stained cells were thenanalyzed by flow cytometry. A total of 10,000 cells was counted for eachsample and divided into four sub populations according to stainingintensity; cells negative for both PI and Annexin (−PI, −Annexin) aretermed live; cells negative for PI and positive for Annexin (−PI,+Annexin) are going through early stages of apoptosis; cells positivefor PI and Annexin (+PI, +Annexin) are dead cells that underwent anapoptotic process; and cells positive for PI and negative for Annexin(+PI, −Annexin) are assumed as dead cells that died by a non-apoptoticprocess such as necrosis.

FIGS. 18A, 18B and 18C illustrate the in vivo effect of the indicatedpeptides in a mouse xenograft model. MDA-MB-231 cells expressingendogenous mutant p53 and stably expressing luciferase were injectedinto the left hip of CD1 nude/nude mice. When tumors reached visiblesize, bioluminescence (indicative of the number of cancer cells) wasmeasured with the IVIS200 system. The mice were then treated byintra-tumoral injection, three times a week, with a mixture of 3 controlpeptides that showed no phenotype in vitro (pCAPs 76, 77 and 12; 2 mg ofeach peptide). 35 days after initiation of treatment, the experiment wasterminated. FIG. 18A shows a logarithmic scale graph demonstrating theluciferase readings in each tumor as a function of time after initiationof treatment (peptide injection). FIG. 18B shows live imaging images ofmice (7-10), at the beginning of treatment. FIG. 18C shows live imagingimages of treated mice (7-9) at day 35, when the experiment wasterminated. Mouse 10 had to be sacrificed after 28 days due to largetumor size.

FIGS. 19A, 19B and 19C illustrate the in vivo effect of the indicatedpeptides in a mouse xenograft model. MDA-MB-231 cells expressingendogenous mutant p53 and stably expressing luciferase were injectedinto the left hip of CD1 nude/nude mice. When tumors reached visiblesize, bioluminescence (indicative of the number of cancer cells) wasmeasured with the IVIS200 system. The mice were then treated byintra-tumoral injection, three times a week, with a mixture of 3 testpeptides that exhibited mutant p53-reactivating ability (pCAPs 159, 155and 174; 2 mg of each peptide). 35 days after initiation of treatment,the experiment was terminated. FIG. 19A shows a logarithmic scale graphdemonstrating the luciferase readings in each tumor as a function oftime after initiation of treatment (peptide injection). FIG. 19B showslive imaging images of mice 1-6 at the beginning of treatment. FIG. 19Cshows live imaging images of treated mice 1-6 at day 35, when theexperiment was terminated. Two of the tumors (mouse 1 and mouse 4)showed a partial response to the treatment, as measured by a decrease of50% and 65%, respectively, in the luciferase signal after 35 days. Mice2 and 5 showed a complete response, reaching bioluminescence readingsthat are as low as or close to the background threshold detection levelsof the IVIS system (5×10⁶ photons) even after 21 days of treatment.Following cessation of the treatment after 35 days, mice numbers 2 and 5were kept alive and monitored for an additional 21 days; no reappearanceof tumors was detected either visually or by live imaging.

FIGS. 20A, 20B, 20C and 20D illustrate the in vivo effect of theindicated peptides in a mouse xenograft model. MDA-MB-231 cellsexpressing endogenous mutant p53 and stably expressing luciferase wereinjected into the left hip of CD1 nude/nude mice. When tumors reachedvisible size, bioluminescence (indicative of the number of cancer cells)was measured with the IVIS200 system. The mice were then treated byintra-tumoral injection, three times a week, with either a mixture of 3control peptides that showed no phenotype in vitro (pCAPs 76, 77 and 12;2 ug of each peptide) or a mixture of 3 test peptides that exhibitedmutant p53-reactivating ability (pCAPs 159, 155 and 174; 2 ug of eachpeptide). FIGS. 20A and 20B show a logarithmic scale graph demonstratingthe average luciferase readings in tumors as a function of time, before(until day 18) and after initiation of treatment (peptide injection).FIGS. 20C and 20D show live imaging images of mice, at the beginning oftreatment (day 18, left) and 12 days into treatment (day 30, right). 40%of mice showed a complete response, reaching bioluminescence readingsthat are as low as or close to the background threshold detection levelsof the IVIS system (5×10⁶ photons).

FIGS. 21A, 21B, 21C, 21D and 21E illustrate the in vivo effect of theindicated peptides in a mouse xenograft model. SW-480 colon cancer cellsexpressing endogenous mutant p53 and stably expressing luciferase wereinjected into the left hip of CD1 nude/nude mice. When tumors reachedvisible size, bioluminescence (indicative of the number of cancer cells)was measured with the IVIS200 system. The mice were then treated byintra-tumoral injection, three times a week, with either a mixture of 3control peptides that showed no phenotype in vitro (pCAPs 76, 77 and 12;2 ug of each peptide) or a mixture of 3 test peptides that exhibitedmutant p53-reactivating ability (pCAPs 250, 308 and 325; 2 ug of eachpeptide). FIGS. 21A, 21B and 21C show a logarithmic scale graphdemonstrating the average luciferase readings in tumors as a function oftime, before (until day 0) and after initiation of treatment (peptideinjection). FIGS. 21D and 21E shows box plot of tumors volume and tumorweight, respectively. As seen in FIGS. 21D and 21E tumors extracted frommice treated with either peptide mix or the pCAP-325 single peptide, aresignificantly smaller in size and weight compared to tumors extractedfrom mice treated with the control peptides (p-value <0.05).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides highly potent peptides and modifiedpeptide agents that can efficiently reactivate p53 conformationalmutants, ideally by changing the mutant p53 proteins conformation and/oractivity to resemble that of a wild type, functional p53 protein. Thepresent invention thus provides peptides and their use in treatingmutant p53 related conditions, where activation of present yetconformationally defective p53 proteins may be beneficial.

The present invention is based on the surprising identification ofhighly potent peptide and peptide-based agents that can efficientlyreactivate p53 conformational mutants, more efficiently than previouslyknown peptides identified for this use.

The present invention provides agents capable of at least partlyelevating the anti-cancer and/or pro-apoptotic effect of mutant p53proteins, and their use in treatment of any disease or condition causedby, or correlated with, a conformationally-aberrant p53 protein. Withoutbeing bound to any mechanism or theory, it is speculated that theconformational change in mutant p53 proteins upon binding to the agentsprovided by the present invention brings them closer to a 3Dconformation of a wild type p53 protein, and thus at least partlyrestores at least part of the functions of a wild type p53 protein tothe mutant p53 proteins.

More specifically, the present invention provides, in an aspect, arecombinant or synthetic peptide consisting of the amino-acid sequenceset forth in any one of SEQ ID NOs:321-286.

The present invention further provides, in another aspect, a recombinantor synthetic peptide comprising the amino-acid sequence set forth in anyone of SEQ ID NOs:321-286, wherein the peptide at least partiallyreactivates a mutant p53 protein.

The present invention further provides, in yet another aspect, arecombinant or synthetic peptide comprising a consensus motif of theamino-acid sequence set forth in any one of SEQ ID NOs:314, 268, 282,340, 376, 298, 377, 378, 253, 20, 379, 302, 275, 380, 273, 381, 280 or382, wherein the peptide at least partially reactivates a mutant p53protein.

In certain embodiments, the peptide consists of the amino-acid sequenceset forth in any one of SEQ ID NO:321, SEQ ID NO:314, SEQ ID NO:313, SEQID NO:310 or SEQ ID NO:307. Each possibility represents a separateembodiment of the invention. In certain embodiments, the peptidedescribed above consists the amino-acid sequence set forth in any one ofSEQ ID NOs:321-302. Each possibility represents a separate embodiment ofthe invention. In certain embodiments, the peptide described aboveconsists the amino-acid sequence set forth in any one of SEQ IDNOs:321-312. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the peptide described above consiststhe amino-acid sequence set forth in any one of SEQ ID NOs:321-316. Eachpossibility represents a separate embodiment of the invention.

In certain embodiments, the peptide comprises of the amino-acid sequenceset forth in any one of SEQ ID NO:321, SEQ ID NO:314, SEQ ID NO:313, SEQID NO:310 or SEQ ID NO:307. Each possibility represents a separateembodiment of the invention. In certain embodiments, the peptidedescribed above comprises the amino-acid sequence set forth in any oneof SEQ ID NOs:321-302. In certain embodiments, the peptide describedabove comprises the amino-acid sequence set forth in any one of SEQ IDNOs:321-312. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the peptide described above comprisesthe amino-acid sequence set forth in any one of SEQ ID NOs:321-316. Eachpossibility represents a separate embodiment of the invention.

The present invention further provides, in yet another aspect, arecombinant or synthetic peptide comprising the amino-acid sequence setforth in any one of SEQ ID NOs: 307, 310, 313, 314 and 321, wherein saidpeptide at least partially reactivates a mutant p53 protein.

The present invention further provides, in yet another aspect, arecombinant or synthetic peptide comprising the amino-acid sequence setforth in any one of SEQ ID NOs: 312, 314, 315, 316, 318 and 321, whereinsaid peptide at least partially reactivates a mutant p53 protein. Eachpossibility represents a separate embodiment of the invention.

According to a specific embodiment the consensus motif is as set forthin SEQ ID NO: 314.

In certain embodiments, the peptide consists of the amino-acid sequenceset forth in any one of SEQ ID NOs: 307, 310, 313, 314 and 321, whereinsaid peptide at least partially reactivates a mutant p53 protein. Eachpossibility represents a separate embodiment of the invention.

In certain embodiments, the peptide consists of the amino-acid sequenceset forth in any one of SEQ ID NOs: 312, 314, 315, 316, 318 and 321,wherein said peptide at least partially reactivates a mutant p53protein. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the peptide the peptide comprises SEQ ID NOs:307, 310, 313, 314 and 321. Each possibility represents a separateembodiment of the invention.

In certain embodiments, the peptide comprises the amino-acid sequenceset forth in SEQ ID NOs:321-316. Each possibility represents a separateembodiment of the invention.

In certain embodiments, the peptide is conjugated to at least one fattyacid moiety. In certain embodiments, the fatty acid is selected from thegroup consisting of myristic acid, lauric acid, palmitic acid andstearic acid. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the fatty acid is a myristoyl fattyacid.

In certain embodiments, the peptide at least partially changes theconformation of the mutant p53 protein to a conformation of a wild-type(WT) p53 protein.

Known in the art are antibodies that specifically recognize only wildtype p53 proteins. Such antibodies are highly useful in determiningwhether a certain p53 protein, either wild type or mutant, holds theconformation of a wild type, functional p53 protein. Thus, in certainembodiments, the peptide at least partially changes the conformation ofthe mutant p53 protein such that the mutant p53 protein is recognized bya monoclonal antibody exclusively directed against a WT p53 protein oragainst a p53 protein holding a WT p53 protein conformation. In certainembodiments, the monoclonal antibody is Ab1620.

It should be understood that since p53 is expressed from both alleles,the overall content of intra-cellular p53 can be either wild-type(wt/wt), mixture of wt and mutant p53 (wt/mut) or mutant p53 only (whenboth alleles are mutated (mut/mut), or one allele is deleted (mut/−)).In cancer, the situation is often wt/mut, mut/mut or mut/−. Since p53acts as a tetramer, mutant p53 proteins may abrogate the activity ofwild type p53 proteins, which may exist in the cancer's cells.Therefore, the peptides provided by the present invention areparticularly useful in treating cancers in which increasing the level ofwild type p53 proteins is not fruitful.

In certain embodiments, the peptide at least partially restores theactivity of the mutant p53 protein to at least one of the activities ofa WT p53 protein.

As used herein the term “reducing” refers to statistically significantlydecreasing a certain phenotype by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%75%, 80%, 95% or even 100% as compared to a control (e.g.,same cell/animal system treated with a control vehicle or non-treated atall) under the same assay conditions.

As used herein the term “increasing” or “improving” refers tostatistically significantly increasing a certain phenotype by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%75%, 80%, 95% or even 100% ascompared to a control (e.g., same cell/animal system treated with acontrol vehicle or non-treated at all) under the same assay conditions.

In certain embodiments, the activity is reducing viability of cellsexpressing the mutant p53 protein. In certain embodiments, the activityis promoting apoptosis of cells expressing the mutant p53 protein. Incertain embodiments, the activity is activating pro-apoptotic genes ofcells expressing said mutant p53 protein. In certain embodiments, thepro-apoptotic genes are selected from the group consisting of CD95, Bax,DR4, DR5, PUMA, NOXA, Bid, 53AIP1 and PERP. Each possibility representsa separate embodiment of the invention.

In certain embodiments, the activity is binding to a p53 consensus DNAbinding element in cells expressing the mutant p53 protein. In certainembodiments, the consensus DNA binding element comprises or consists theamino-acid sequence set forth in SEQ ID NO:339.

Methods of monitoring cellular changes induced by the any of thepeptides of the present invention are known in the art and include forexample, the MTT test which is based on the selective ability of livingcells to reduce the yellow salt MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (Sigma,Aldrich St Louis, Mo., USA) to a purple-blue insoluble formazanprecipitate; the BrDu assay [Cell Proliferation ELISA BrdU colorimetrickit (Roche, Mannheim, Germany]; the TUNEL assay [Roche, Mannheim,Germany]; the Annexin V assay [ApoAlert® Annexin V Apoptosis Kit(Clontech Laboratories, Inc., CA, USA)]; the Senescenceassociated-β-galactosidase assay (Dimri G P, Lee X, et al. 1995. Abiomarker that identifies senescent human cells in culture and in agingskin in vivo. Proc Natl Acad Sci USA 92:9363-9367); as well as variousRNA and protein detection methods (which detect level of expressionand/or activity) which are further described hereinbelow.

In certain embodiments, the binding results in at least partialactivation of an endogenous p53 target gene. In certain embodiments, theendogenous target gene is selected from the group consisting of p21,MDM2 and PUMA. Each possibility represents a separate embodiment of theinvention.

In certain embodiments, the mutant p53 protein is of a differentconformation than a WT p53 protein. In certain embodiments, the mutantp53 protein is at least partly inactive compared to a WT p53 protein.

In certain embodiments, the mutant p53 protein is not recognized by amonoclonal antibody directed against a WT p53 protein. In certainembodiments, the mutant p53 protein, upon binding to the peptide, isrecognized by a monoclonal antibody directed against a WT p53 protein.In certain embodiments, the monoclonal antibody is Ab1620.

In certain embodiments, the mutant p53 protein comprises a mutationselected from the group consisting of R175H, V143A, R249S, R273H, R280K,P309S, P151S, P151H, C176S, C176F, H179L, Q192R, R213Q, Y220C, Y220D,R245S, R282W, D281G, S241F, C242R, R248Q, R248W, D281G, R273C and V274F.Each possibility represents a separate embodiment of the invention.

In certain embodiments, the peptide comprises the consensus motif setforth in SEQ ID NO:314. In certain embodiments, the peptide comprisesthe amino-acid sequence set forth in any one of SEQ ID NO:321, SEQ IDNO:314, SEQ ID NO:313, SEQ ID NO:310 or SEQ ID NO:307. Each possibilityrepresents a separate embodiment of the invention. In certainembodiments, the peptide consists of the amino-acid sequence set forthin any one of SEQ ID NO:321, SEQ ID NO:314, SEQ ID NO:313, SEQ ID NO:310or SEQ ID NO:307. Each possibility represents a separate embodiment ofthe invention. In certain embodiments, the peptide comprises theamino-acid sequence set forth in any one of SEQ ID NOs:268, 282, 340,376, 298, 377, 378, 253, 20, 379, 302, 275, 380, 273, 381, 280 or 382.Each possibility represents a separate embodiment of the invention. Incertain embodiments, the peptide comprises the amino-acid sequence setforth in any one of SEQ ID NOs:379, 302, 275, 380, 273, 381, 280 or 382.Each possibility represents a separate embodiment of the invention. Incertain embodiments, the peptide comprises the amino-acid sequence setforth in any one of SEQ ID NOs:302, 275, 380, 273, 381, 280 or 382. Eachpossibility represents a separate embodiment of the invention. Accordingto a specific embodiment the consensus motif is as set forth in SEQ IDNO: 314. In certain embodiments, the peptide consists of the amino-acidsequence set forth in any one of SEQ ID NOs: 307, 310, 313, 314 and 321,wherein said peptide at least partially reactivates a mutant p53protein. Each possibility represents a separate embodiment of theinvention. In certain embodiments, the peptide consists of theamino-acid sequence set forth in any one of SEQ ID NOs: 312, 314, 315,316, 318 and 321, wherein said peptide at least partially reactivates amutant p53 protein. Each possibility represents a separate embodiment ofthe invention. In certain embodiments, the peptide the peptide comprisesSEQ ID NOs: 307, 310, 313, 314 and 321. Each possibility represents aseparate embodiment of the invention. In certain embodiments, thepeptide comprises the amino-acid sequence set forth in SEQ IDNOs:321-316. Each possibility represents a separate embodiment of theinvention.

The present invention further provides, in another aspect, an expressionvector, capable of expressing the peptides described above.

The present invention further provides, in another aspect, apharmaceutical composition, comprising the peptides described above.

The present invention further provides, in yet another aspect, apharmaceutical composition, comprising the expression vector describedabove.

In an aspect, the pharmaceutical compositions described above are foruse in treating a disease, disorder or condition associated with amutant p53 protein.

In some embodiments, the disease is cancer. In some embodiments, thecancer is selected from the group consisting of breast cancer, coloncancer and lung cancer. Each possibility represents a separateembodiment of the invention. In some embodiments, the cancer cellsexpress the mutant p53 protein.

The present invention further provides, in another aspect, a method oftreating a disease, disorder or condition associated with a mutant p53protein, comprising the step of administering a therapeuticallyeffective amount of the pharmaceutical compositions described above to asubject in need thereof, thereby treating the disease, disorder orcondition.

The present invention further provides, in yet another aspect, a kitcomprising the pharmaceutical compositions described above.

In an aspect, the kit described above is for use in treating a disease,disorder or condition associated with a mutant p53 protein.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below. It is to be understood that theseterms and phrases are for the purpose of description and not oflimitation, such that the terminology or phraseology of the presentspecification is to be interpreted by the skilled artisan in light ofthe teachings and guidance presented herein, in combination with theknowledge of one of ordinary skill in the art.

The term “recombinant or synthetic peptide” as used herein refers to apeptide produced by standard biotechnological methods known in the art,such as expression in bacteria or Solid-phase peptide synthesis (SPPS).

The term “capable of at least partially reactivating a mutant p53protein” or “at least partially reactivates a mutant p53 protein” asinterchangeably used herein refers to peptide, wherein upon binding ofthe peptide to a mutant p53 protein, the mutant p53 protein gains orincreases an activity similar to a corresponding activity of a wild typep53 protein.

The term “consensus motif′ as used herein refers to an amino acidsequence of at least three amino acids, which was found in more than onepeptide provided by the present invention.

As used herein the phrase “permeability-enhancing moiety” refers to anagent which enhances translocation of any of the attached peptide acrossa cell membrane.

Any moiety known in the art to facilitate actively or passively orenhance permeability of compositions into cells may be used forconjugation with the peptide core according to the present invention.Non-limitative examples include: hydrophobic moieties such as fattyacids, steroids and bulky aromatic or aliphatic compounds; moietieswhich may have cell-membrane receptors or carriers, such as steroids,vitamins and sugars, natural and non-natural amino acids andproteinaceous moiety e.g., transporter peptides, also referred to as“cell penetrating peptides” or a CPP, poly-Arginine or poly-Lysine, acombination of same or an antibody. According to some embodiments, theproteinaceous moiety is a CPP.

According to some embodiments, the proteinaceous moiety ispoly-Arginine.

According to some embodiments, the hydrophobic moiety is a lipid moietyor an amino acid moiety.

Cell-Penetrating Peptides (CPPs) are short peptides (≦40 amino acids),with the ability to gain access to the interior of almost any cell. Theyare highly cationic and usually rich in arginine and lysine amino acids.Indeed the present inventors have used positively charged amino acids(on either peptide termini) or poly-cationic amino acids (at least 2e.g., 2-12) poly-Arg to impart the peptides with cell permeation. Theyhave the exceptional property of carrying into the cells a wide varietyof covalently and noncovalently conjugated cargoes such as proteins,oligonucleotides, and even 200 nm liposomes. Therefore, according toadditional exemplary embodiment CPPs can be used to transport thepeptides to the interior of cells.

TAT (transcription activator from HIV-1), pAntp (also named penetratin,Drosophila antennapedia homeodomain transcription factor) and VP22 (fromHerpes Simplex virus) are examples of CPPs that can enter cells in anon-toxic and efficient manner and may be suitable for use with someembodiments of the invention. Protocols for producing CPPs-cargosconjugates and for infecting cells with such conjugates can be found,for example L Theodore et al. [The Journal of Neuroscience, (1995)15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994)91:664-668], and Jing Bian et al. [Circulation Research (2007) 100:1626-1633].

However, the disclosure is not so limited, and any suitable penetratingagent may be used, as known by those of skill in the art.

When the peptides of the present invention are attached to cellpenetrating peptides, it is contemplated that the full length peptide isno greater than 30 amino acids, no greater than 25 amino acids, nogreater than 22 amino acids, no greater than 20 amino acids, no greaterthan 15 amino acids, no greater than 12 amino acids, no greater than 10amino acids, no greater than 9 amino acids, no greater than 8 aminoacids, or no greater than 7 amino acids.

The term “fatty acid moiety” as used herein refers to a part of a fattyacid that exhibits a particular set of chemical and pharmacologiccharacteristics similar to the corresponding complete fatty acid originmolecule. The term further refers to any molecular species and/ormolecular fragment comprising the acyl component of a fatty (carboxylic)acid.

A permeability-enhancing moiety according to the present invention ispreferably connected covalently to the peptide sequence via a directbond or via a linker, to form a peptide conjugate. Thepermeability-enhancing moiety may be connected to any position in thepeptide moiety, directly or through a spacer, preferably to the aminoterminus of the peptide. According to certain embodiments, thepermeability enhancing moiety is a fatty acid.

The term “Permeability” as used herein refers to the ability of an agentor substance to penetrate, pervade, or diffuse through a barrier,membrane, or a skin layer. A “cell permeability” or a “cell-penetration”moiety refers to any molecule known in the art which is able tofacilitate or enhance penetration of molecules through membranes.Non-limitative examples include: hydrophobic moieties such as lipids,fatty acids, steroids and bulky aromatic or aliphatic compounds;moieties which may have cell-membrane receptors or carriers, such assteroids, vitamins and sugars, natural and non-natural amino acids,transporter peptides, nanoparticles and liposomes.

The hydrophobic moiety according to the invention may preferablycomprise a lipid moiety or an amino acid moiety. According to a specificembodiment the hydrophobic moiety is selected from the group consistingof: phospholipids, steroids, sphingosines, ceramides, octyl-glycine,2-cyclohexylalanine, benzolylphenylalanine, propionoyl (C₃); butanoyl(C₄); pentanoyl (C₅); caproyl (C₆); heptanoyl (C₇); capryloyl (C₈);nonanoyl (C₉); capryl (C₁₀); undecanoyl (C₁₁); lauroyl (C₁₂);tridecanoyl (C₁₃); myristoyl (C₁₄); pentadecanoyl (C₁₅); palmitoyl(C₁₆); phtanoyl ((CH₃)₄); heptadecanoyl (C₁₇); stearoyl (C₁₈);nonadecanoyl (C₁₉); arachidoyl (C₂₀); heniecosanoyl (C₂₁); behenoyl(C₂₂); trucisanoyl (C₂₃); and lignoceroyl (C₂₄); wherein saidhydrophobic moiety is attached to said chimeric polypeptide with amidebonds, sulfhydryls, amines, alcohols, phenolic groups, or carbon-carbonbonds.

Other examples for lipidic moieties which may be used according to thepresent invention: Lipofectamine, Transfectace, Transfectam, Cytofectin,DMRIE, DLRIE, GAP-DLRIE, DOTAP, DOPE, DMEAP, DODMP, DOPC, DDAB, DOSPA,EDLPC, EDMPC, DPH, TMADPH, CTAB, lysyl-PE, DC-Cho, -alanyl cholesterol;DCGS, DPPES, DCPE, DMAP, DMPE, DOGS, DOHME, DPEPC, Pluronic, Tween,BRIJ, plasmalogen, phosphatidylethanolamine, phosphatidylcholine,glycerol-3-ethylphosphatidylcholine, dimethyl ammonium propane,trimethyl ammonium propane, diethylammonium propane, triethylammoniumpropane, dimethyldioctadecylammonium bromide, a sphingolipid,sphingomyelin, a lysolipid, a glycolipid, a sulfatide, aglycosphingolipid, cholesterol, cholesterol ester, cholesterol salt,oil, N-succinyldioleoylphosphatidylethanolamine,1,2-dioleoyl-sn-glycerol, 1,3-dipalmitoyl-2-succinylglycerol,1,2-dipalmitoyl-sn-3-succinylglycerol,1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine,palmitoylhomocystiene,N,N′-Bis(dodecyaminocarbonylmethylene)-N,N′-bis((-N,N,N-trimethylammoniumethyl-aminocarbonylmethylene)ethylenediamine tetraiodide;N,N″-Bis(hexadecylaminocarbonylmethylene)-N,N′,N″-tris((-N,N,N-trimethylammonium-ethylaminocarbonylmethylenediethylenetriaminehexaiodide;N,N′-Bis(dodecylaminocarbonylmethylene)-N,N″-bis((-N,N,N-trimethylammoniumethylaminocarbonylmethylene)cyclohexylene-1,4-diamine tetraiodide;1,7,7-tetra-((-N,N,N,N-tetramethylammoniumethylamino-carbonylmethylene)-3-hexadecylaminocarbonyl-methylene-1,3,7-triaazaheptaneheptaiodide;N,N,N′,N′-tetra((-N,N,N-trimethylammonium-ethylaminocarbonylmethylene)-N′-(1,2-dioleoylglycero-3-phosphoethanolaminocarbonylmethylene)diethylenetriam ine tetraiodide;dioleoylphosphatidylethanolamine, a fatty acid, a lysolipid,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, a sphingolipid, aglycolipid, a glucolipid, a sulfatide, a glycosphingolipid, phosphatidicacid, palmitic acid, stearic acid, arachidonic acid, oleic acid, a lipidbearing a polymer, a lipid bearing a sulfonated saccharide, cholesterol,tocopherol hemisuccinate, a lipid with an ether-linked fatty acid, alipid with an ester-linked fatty acid, a polymerized lipid, diacetylphosphate, stearylamine, cardiolipin, a phospholipid with a fatty acidof 6-8 carbons in length, a phospholipid with asymmetric acyl chains,6-(5-cholesten-3b-yloxy)-1-thio-b-D-galactopyrano side,digalactosyldiglyceride,6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxy-1-thio-b-D-galactopyranoside,6-(5-cholesten-3b-yloxy)hexyl-6-amino-6-deoxyl-1-thio-a-D-mannopyranoside,12-(((7′-diethylamino-coumarin-3-yl)carbonyl)methylamino)-octadecanoicacid; N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadecanoyl]-2-aminopalmitic acid;cholesteryl)4′-trimethyl-ammonio)butanoate;N-succinyldioleoyl-phosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinyl-glycerol;1,3-dipalmitoyl-2-succinylglycerol,1-hexadecyl-2-palmitoylglycero-phosphoethanolamine, andpalmitoylhomocysteine.

According to a specific embodiment, the p53 protein is human p53.According to a specific embodiment, the subject, the biological samplederived therefrom or the cell (as described below) are of a human being.

The term “cells expressing the mutant p53 protein” as used herein refersto cells which express from at least one allele a mutant p53 protein. Incertain embodiments, the term “cells expressing the mutant p53 protein”is interchangeable with “cancer cells”.

The term “pro-apoptotic genes” refers to a gene, or a multitude ofgenes, involved in apoptosis, either directly (such as certain caspases)or indirectly (for example, as part of a signal transduction cascade).

The term “pharmaceutical composition” as used herein refers to anycomposition comprising at least one pharmaceutically active ingredient.

The term “associated with a mutant p53 protein” as used herein refers toany disease, disorder or condition which is caused by a mutant p53protein or related to the presence of a mutant p53 protein in a cell oran organ.

It should be understood that since p53 is expressed from both alleles,the overall content of intra-cellular p53 can be either wild-type(wt/wt), mixture of wt and mutant p53 (wt/mut) or mutant p53 only (whenboth alleles are mutated (mut/mut), or one allele is deleted (mut/−)).In cancer, the situation is often wt/mut, mut/mut or mut/−. Since p53acts as a tetramer, mutant p53 proteins may abrogate the activity ofwild type p53 proteins, which do exist in the cancer's cells. Therefore,the peptides provided by the present invention are particularly usefulin treating cancers in which increasing the level of wild type p53proteins is not fruitful.

The term “therapeutically effective amount” as used herein refers to anamount of a composition containing a peptide according to the presentinvention that is sufficient to reduce, decrease, and/or inhibit adisease, disorder or condition in an individual.

As used herein, the term p53 is directed to a p53 protein that can havea conformation of a WT p53, a mutated p53, or an intermediateconformation between WT and mutated p53.

As used herein, the terms “wild type p53”, “wt p53” and “WT p53” mayinterchangeably be used and are directed to a wild type p53 protein,having the conformation of a wild type p53 protein and hence, activityof a wild type p53 protein. In some embodiments, wild type p53 can beidentified by a specific monoclonal antibody.

As used herein, the terms “mutant p53”, “Mut-p53”, “mutated p53”, and“p53 mutant” may interchangeably be used and are directed to a mutatedp53 protein, incapable of efficiently functioning in a target cell. Insome embodiments, a Mut-p53 cannot bind its target site. In someembodiments, a Mut-p53 is mutated at the DNA binding domain (DBD)region. In some embodiments, a Mut-p53 is misfolded in an inactiveconformation. In some exemplary embodiments, the Mut-p53 is atemperature sensitive (ts) mut p53 R249S (R249S p53), a hot spot fulllength mutant p53 Mut-p53 R175H (R175H p53), or any other Mut-p53protein. In some embodiments, a Mut-p53 is identified by a specificmonoclonal antibody, capable of recognizing a misfolded conformation ofp53 (induced by the mutation of the p53). In some embodiments, a Mut-p53is identified by a specific monoclonal antibody.

The phrase “peptide reactivates a mutant p53 protein” as used hereinrefers to a peptide which upon its interaction with a mutant p53protein, the mutant p53 protein increases at least one of hisactivities, wherein the activities are the activities of a wild type p53protein. For example, upon its interaction with a peptide provided bythe present invention, a mutant p53 protein may increase, directly orindirectly, the expression of pro-apoptotic proteins such as caspases ina cancer cell, in a similar way to what would a wild type p53 protein doin a similar situation.

As referred to herein, the terms “reactivating peptide”, “Mut-p53reactivating peptide” or “the peptide” may interchangeably be used andare directed to a peptidic agent capable of at least partially restoringactivity to Mut-p53. In some embodiments, the reactivating agent canreactivate a Mut-p53 by affecting the conformation of the Mut-p53, toassume a conformation which is more similar to or identical to a native,WT p53. In some embodiments, the reactivating agent can reactivate aMut-p53 to restore binding of the Mut-p53 to a WT p53 binding site in atarget DNA. In some embodiments, the reactivating agent can restorebiochemical properties of the Mut-p53. In some embodiments, thereactivating agent can induce the Mut-p53 protein to exhibitp53-selective inhibition of cancer cells. In some embodiments, thereactivating agent can reactivate a Mut-p53 to have structuralproperties, biochemical properties, physiological properties and/orfunctional properties similar to or identical to a WT p53 protein. Insome embodiments, the reactivating agent is a peptide. In someembodiments, the reactivating agent is a peptide having 3-30 amino acidsin length. In some embodiments, the reactivating agent is a peptidehaving 7-30 amino acids in length. In some embodiments, the reactivatingagent is a peptide having 12-30 amino acids in length. In someembodiments, the reactivating agent is a peptide having 3-25 amino acidsin length. In some embodiments, the reactivating agent is a peptidehaving 7-25 amino acids in length. In some embodiments, the reactivatingagent is a peptide having 12-25 amino acids in length. In someembodiments, the reactivating agent is a peptide having 3-22 amino acidsin length. In some embodiments, the reactivating agent is a peptidehaving 7-22 amino acids in length. In some embodiments, the reactivatingagent is a peptide having 12-22 amino acids in length. In someembodiments, the reactivating agent is a peptide having 7-9 amino acidsin length. In some embodiments, the reactivating agent is a peptidehaving 6-9 amino acids in length. In some embodiments, the reactivatingagent is a peptide having 7-10 amino acids in length. In someembodiments, the reactivating agent is a peptide having 6-10 amino acidsin length. In some embodiments, the reactivating agent is a peptidehaving 5-20 amino acids in length. In some embodiments, the reactivatingagent is a peptide having 6-15 amino acids in length. In someembodiments, the reactivating agent is a peptide having 7 or 12 aminoacids in length.

The term “conformation” with respect to a protein is directed to thestructural arrangement (folding) of a protein in space.

The terms “deep sequencing” and “next generation sequencing” mayinterchangeably be used and are directed to an enhanced sequencingmethod enabling the rapid parallel sequencing of multiple nucleic acidsequences.

The “phage display” method includes the screening of a library ofphages, each expressing and presenting a specific, exogenous molecule,such as a peptide. The enrichment of phages that express and present aspecific peptide is achieved by affinity selection of a phage library onimmobilized target. In this “panning” process, binding phages (i.e.phages which express and present a peptide that can bind the immobilizedtarget) are captured, whereas nonbinding phages (i.e., phages which donot express and present a peptide that can bind the immobilized target)are washed off. A next step in the method can include the elution andamplification of the bound phages by reinfection of E. coli cells withthe identified phages. In some embodiments, a phage library can be anoriginal library, or a commercially available phage display library.

The terms “polypeptide” and “peptide” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers.

The term “peptide” as used herein encompasses native peptides (eitherdegradation products, synthetically synthesized peptides or recombinantpeptides) and peptidomimetics (typically, synthetically synthesizedpeptides), as well as peptoids and semipeptoids which are peptideanalogs, which may have, for example, modifications rendering thepeptides more stable while in a body or more capable of penetrating intocells. Such modifications include, but are not limited to N terminusmodification, C terminus modification, peptide bond modification,backbone modifications, and residue modification. Methods for preparingpeptidomimetic compounds are well known in the art and are specified,for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as if fully set forth herein. Further details in this respectare provided hereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated amide bonds (—N(CH3)-CO—), ester bonds(—C(═O)—O—), ketomethylene bonds (—CO—CH2-), sulfinylmethylene bonds(—S(═O)—CH2-), α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl (e.g.,methyl), amine bonds (—CH2-NH—), sulfide bonds (—CH2-S—), ethylene bonds(—CH2-CH2-), hydroxyethylene bonds (—CH(OH)-CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), fluorinated olefinic doublebonds (—CF═CH—), retro amide bonds (—NH—CO—), peptide derivatives(—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturally presenton the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) bonds at the same time.

“Conservative substitution” refers to the substitution of an amino acidin one class by an amino acid of the same class, where a class isdefined by common physico-chemical amino acid side chain properties andhigh substitution frequencies in homologous proteins found in nature, asdetermined, for example, by a standard Dayhoff frequency exchange matrixor BLOSUM matrix. Six general classes of amino acid side chains havebeen categorized and include: Class I (Cys); Class II (Ser, Thr, Pro,Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys);Class V (He, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example,substitution of an Asp for another Class III residue such as Asn, Gin,or Glu, is a conservative substitution.

Other classifications include positive amino acids (Arg, His, Lys),negative amino acids (Asp, Glu), polar uncharged (Ser, Thr, Asn, Gln),hydrophobic side chains (Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp).

pCap 250 (SEQ ID NO: 321) comprising the core sequence of HSTPHP (SEQ IDNO: 314), may be conservatively modified to include any of the aboveamino acid conservative substitutions, wherein each option is consideredas a separate embodiment.

“Non-conservative substitution” refers to the substitution of an aminoacid in one class with an amino acid from another class; for example,substitution of an Ala, a Class II residue, with a Class III residuesuch as Asp, Asn, Glu, or Gin.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted bynon-natural aromatic amino acids such as1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine,ring-methylated derivatives of Phe, halogenated derivatives of Phe orO-methyl-Tyr. Other synthetic options are listed hereinbelow in Table B.

The peptides of some embodiments of the invention may also include oneor more modified amino acids or one or more non-amino acid monomers(e.g. fatty acids, complex carbohydrates etc).

The term “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Tables A and B below list naturally occurring amino acids (Table A), andnon-conventional or modified amino acids (e.g., synthetic, Table B)which can be used with some embodiments of the invention.

TABLE A Three-Letter One-letter Amino Acid Abbreviation Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid as above XaaX

TABLE B Non-conventional amino acid Code Non-conventional amino acidCode ornithine Orn hydroxyproline Hyp α-aminobutyric acid Abuaminonorbornyl- Norb carboxylate D-alanine Dala aminocyclopropane- Cprocarboxylate D-arginine Darg N-(3-guanidinopropyl)glycine NargD-asparagine Dasn N-(carbamylmethyl)glycine Nasn D-aspartic acid DaspN-(carboxymethyl)glycine Nasp D-cysteine Dcys N-(thiomethyl)glycine NcysD-glutamine Dgln N-(2-carbamylethyl)glycine Ngln D-glutamic acid DgluN-(2-carboxyethyl)glycine Nglu D-histidine DhisN-(imidazolylethyl)glycine Nhis D-isoleucine DileN-(1-methylpropyl)glycine Nile D-leucine Dleu N-(2-methylpropyl)glycineNleu D-lysine Dlys N-(4-aminobutyl)glycine Nlys D-methionine DmetN-(2-methylthioethyl)glycine Nmet D-ornithine DornN-(3-aminopropyl)glycine Norn D-phenylalanine Dphe N-benzylglycine NpheD-proline Dpro N-(hydroxymethyl)glycine Nser D-serine DserN-(1-hydroxyethyl)glycine Nthr D-threonine Dthr N-(3-indolylethyl)glycine Nhtrp D-tryptophan Dtrp N-(p-hydroxyphenyl)glycine NtyrD-tyrosine Dtyr N-(1-methylethyl)glycine Nval D-valine DvalN-methylglycine Nmgly D-N-methylalanine Dnmala L-N-methylalanine NmalaD-N-methylarginine Dnmarg L-N-methylarginine Nmarg D-N-methylasparagineDnmasn L-N-methylasparagine Nmasn D-N-methylasparatate DnmaspL-N-methylaspartic acid Nmasp D-N-methylcysteine DnmcysL-N-methylcysteine Nmcys D-N-methylglutamine Dnmgln L-N-methylglutamineNmgln D-N-methylglutamate Dnmglu L-N-methylglutamic acid NmgluD-N-methylhistidine Dnmhis L-N-methylhistidine NmhisD-N-methylisoleucine Dnmile L-N-methylisolleucine NmileD-N-methylleucine Dnmleu L-N-methylleucine Nmleu D-N-methyllysine DnmlysL-N-methyllysine Nmlys D-N-methylmethionine Dnmmet L-N-methylmethionineNmmet D-N-methylornithine Dnmorn L-N-methylornithine NmornD-N-methylphenylalanine Dnmphe L-N-methylphenylalanine NmpheD-N-methylproline Dnmpro L-N-methylproline Nmpro D-N-methylserine DnmserL-N-methylserine Nmser D-N-methylthreonine Dnmthr L-N-methylthreonineNmthr D-N-methyltryptophan Dnmtrp L-N-methyltryptophan NmtrpD-N-methyltyrosine Dnmtyr L-N-methyltyrosine Nmtyr D-N-methylvalineDnmval L-N-methylvaline Nmval L-norleucine Nle L-N-methylnorleucineNmnle L-norvaline Nva L-N-methylnorvaline Nmnva L-ethylglycine EtgL-N-methyl-ethylglycine Nmetg L-t-butylglycine TbugL-N-methyl-t-butylglycine Nmtbug L-homophenylalanine HpheL-N-methyl-homophenylalanine Nmhphe α-naphthylalanine AnapN-methyl-α-naphthylalanine Nmanap penicillamine PenN-methylpenicillamine Nmpen γ-aminobutyric acid GabuN-methyl-γ-aminobutyrate Nmgabu cyclohexylalanine ChexaN-methyl-cyclohexylalanine Nmchexa cyclopentylalanine CpenN-methyl-cyclopentylalanine Nmcpen α-amino-α-methylbutyrate AabuN-methyl-α-amino-α- Nmaabu methylbutyrate α-aminoisobutyric acid AibN-methyl-α-aminoisobutyrate Nmaib D-α-methylarginine DmargL-α-methylarginine Marg D-α-methylasparagine Dmasn L-α-methylasparagineMasn D-α-methylaspartate Dmasp L-α-methylaspartate MaspD-α-methylcysteine Dmcys L-α-methylcysteine Mcys D-α-methylglutamineDmgln L-α-methylglutamine Mgln D-α-methyl glutamic acid DmgluL-α-methylglutamate Mglu D-α-methylhistidine Dmhis L-α-methylhistidineMhis D-α-methylisoleucine Dmile L-α-methylisoleucine MileD-α-methylleucine Dmleu L-α-methylleucine Mleu D-α-methyllysine DmlysL-α-methyllysine Mlys D-α-methylmethionine Dmmet L-α-methylmethionineMmet D-α-methylornithine Dmorn L-α-methylornithine MornD-α-methylphenylalanine Dmphe L-α-methylphenylalanine MpheD-α-methylproline Dmpro L-α-methylproline Mpro D-α-methylserine DmserL-α-methylserine Mser D-α-methylthreonine Dmthr L-α-methylthreonine MthrD-α-methyltryptophan Dmtrp L-α-methyltryptophan Mtrp D-α-methyltyrosineDmtyr L-α-methyltyrosine Mtyr D-α-methylvaline Dmval L-α-methylvalineMval N-cyclobutylglycine Ncbut L-α-methylnorvaline MnvaN-cycloheptylglycine Nchep L-α-methylethylglycine MetgN-cyclohexylglycine Nchex L-α-methyl-t-butylglycine MtbugN-cyclodecylglycine Ncdec L-α-methyl-homophenylalanine MhpheN-cyclododecylglycine Ncdod α-methyl-α-naphthylalanine ManapN-cyclooctylglycine Ncoct α-methylpenicillamine MpenN-cyclopropylglycine Ncpro α-methyl-γ-aminobutyrate MgabuN-cycloundecylglycine Ncund α-methyl-cyclohexylalanine MchexaN-(2-aminoethyl)glycine Naeg α-methyl-cyclopentylalanine McpenN-(2,2-diphenylethyl)glycine Nbhm N-(N-(2,2-diphenylethyl) Nnbhmcarbamylmethyl-glycine N-(3,3-diphenylpropyl)glycine NbheN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl-glycine1-carboxy-1-(2,2-diphenyl Nmbc 1,2,3,4-tetrahydroisoquinoline-3- Ticethylamino)cyclopropane carboxylic acid phosphoserine pSerphosphothreonine pThr phosphotyrosine pTyr O-methyl-tyrosine2-aminoadipic acid hydroxylysine

The peptides of some embodiments of the invention are preferablyutilized in a linear form, although it will be appreciated that in caseswhere cyclicization does not severely interfere with peptidecharacteristics, cyclic forms of the peptide can also be utilized.

In order to improve bioavailability, the peptide may comprise at leastone D amino acid.

Alternatively or additionally, the peptide may comprise C-terminalamidation.

Yet alternatively or additionally the peptide may be conjugated tonon-proteinaceous non-toxic moiety such as, but are not limited to,polyethylene glycol (PEG), Polyvinyl pyrrolidone (PVP), poly(styrenecomaleic anhydride) (SMA), and divinyl ether and maleic anhydridecopolymer (DIVEMA).

It will be appreciated that the peptides of the invention can alsoutilize peptide homologues which exhibit the desired activity (e.g.,reactivation of p53 mutants)), also referred to herein as functionalequivalents, whereby the activity of the peptide homologue is determinedaccording to methods known in the art such as described herein. Suchhomologues can be, for example, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 286-321(e.g., 302-321, 312-321, 316-321 e.g., 321 or 314, e.g., 321), asdetermined using the BestFit software.

The terms “nucleic acid”, “polynucleotide”, “oligonucleotide” or “oligo”relates to a single-stranded or double-stranded polymer composed of DNA(Deoxyribonucleic acid) nucleotides, RNA (Ribonucleic acid) nucleotidesor a combination of both types, and may include natural nucleotides,chemically modified nucleotides and synthetic nucleotides.

“Chemically modified” refers to an amino acid that is modified either bynatural processes, or by chemical modification techniques which are wellknown in the art. Among the numerous known modifications, typical, butnot exclusive examples include: acetylation, acylation, amidation,ADP-ribosylation, glycosylation, glycosaminoglycanation, GPI anchorformation, covalent attachment of a lipid or lipid derivative,methylation, myristlyation, pegylation, prenylation, phos-phorylation,ubiqutination, or any similar process.

As referred to herein, the term “treating a disease” or “treating acondition” is directed to administering a composition, which includes atleast one agent, effective to ameliorate symptoms associated with adisease, to lessen the severity or cure the disease, or to prevent thedisease from occurring in a subject. Administration may include anyadministration route. In some embodiments, the disease is a disease thatis caused by or related to the presence of a mutated p53 in a cell,tissue, organ, body, and the like. In some embodiments, the disease iscancer. In some embodiments, the cancer is selected from the groupconsisting of breast cancer, colon cancer and lung cancer. Eachpossibility represents a separate embodiment of the invention. In someembodiments, the subject is a mammal, such as a human. In someembodiments, the subject is a mammal animal. In some embodiments, thesubject is a non-mammal animal.

The term “expression”, as used herein, refers to the production of adesired end-product molecule in a target cell. The end-product moleculemay include, for example an RNA molecule; a peptide or a protein; andthe like; or combinations thereof.

The term “construct”, as used herein refers to an artificially assembledor isolated nucleic acid molecule which may be one or more nucleic acidsequences, wherein the nucleic acid sequences may comprise codingsequences (that is, sequence which encodes an end product), regulatorysequences, non-coding sequences, or any combination thereof. The termconstruct encompasses, for example, vector but should not be seen asbeing limited thereto.

“Expression vector” refers to vectors that have the ability toincorporate and express heterologous nucleic acid fragments (such as,for example, DNA), in a foreign cell. In other words, an expressionvector comprises nucleic acid sequences/fragments (such as DNA, mRNA,tRNA, rRNA), capable of being transcribed. Many prokaryotic andeukaryotic expression vectors are known and/or commercially available.Selection of appropriate expression vectors is within the knowledge ofthose having skill in the art.

The terms “Upstream” and “Downstream”, as used herein refers to arelative position in a nucleotide sequence, such as, for example, a DNAsequence or an RNA sequence. As well known, a nucleotide sequence has a5′ end and a 3′ end, so called for the carbons on the sugar (deoxyriboseor ribose) ring of the nucleotide backbone. Hence, relative to theposition on the nucleotide sequence, the term downstream relates to theregion towards the 3′ end of the sequence. The term upstream relates tothe region towards the 5′ end of the strand.

As used herein, the terms “introducing”, “transfection” or“transfecting” and “infection” or “infecting” may interchangeably beused and refer to the transfer of molecules, such as, for example,nucleic acids, polynucleotide molecules, vectors, and the like into atarget cell(s), and more specifically into the interior of amembrane-enclosed space of a target cell(s). The molecules can be“introduced” into the target cell(s) by any means known to those ofskill in the art, for example as taught by Sambrook et al. MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NewYork (2001), the contents of which are incorporated by reference herein.Means of “introducing” molecules into a cell include, for example, butare not limited to: heat shock, calcium phosphate transfection, PEItransfection, electroporation, lipofection, transfection agent(s),viral-mediated transfer, and the like, or combinations thereof. Thetransfection of the cell may be performed on any type of cell, of anyorigin.

As referred to herein, the term “exogenous gene” is directed to a gene(or any part thereof) which is introduced from the exterior into a cell.In some embodiments, the exogenous gene is inserted in the form of apolynucleotide (for example, DNA, RNA, and the like). In someembodiments, the exogenous gene is capable of being expressed in thecell.

In some embodiments, the exogenous gene is overexpressed within thecell.

As used herein the term “about” in reference to a numerical value statedherein is to be understood as the stated value+/−10%.

In some embodiments, the reactivating peptide can reactivate a Mut-p53to have structural properties, biochemical properties, physiologicalproperties and/or functional properties similar to or identical to a WTp53 protein.

According to some embodiments, there are provided Mut-p53 reactivatingpeptides, wherein the peptides are in the length of about 3-25 aminoacids. In some embodiments, the Mut-p53 reactivating peptides are in thelength of about 4-15 amino acids. In some embodiments, the Mut-p53reactivating peptides are in the length of about 7-12 amino acids.

In some embodiments, the Mut-p53 reactivating peptides are in the lengthof 7 amino acids. In some embodiments, the Mut-p53 reactivating peptidesare in the length of 12 amino acids. Each possibility represents aseparate embodiment of the invention.

Other peptide lengths are recited throughout the application. Eachpossibility represents a separate embodiment of the invention.

In some embodiments, there is provided a Mut-p53 reactivating peptidehaving an amino acid sequence as denoted by any one of the peptidesequences in Tables 6, 7 or 8, herein below.

According to some embodiments, a Mut-p53 reactivating peptide can affectMut-p53 such that it can trans-activates a reporter gene (such asLuciferase) having WT p53 binding element in its promoter. In someembodiments the transactivation of the reporter gene may be performed invitro (for example, in a test tube or well), or in-vivo in a cell,harboring the reporter gene construct.

According to some embodiments, a Mut-p53 reactivating peptide can bindto the DNA binding Domain (DBD) of a mutated p53. In some embodiments,the mutated p53 harbors a mutation in its DNA binding domain (DBD).

In some embodiments, cancer is adrenocortical carcinoma, anal cancer,bladder cancer, brain tumor, brain stem glioma, brain tumor, cerebellarastrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal, pineal tumors, hypothalamicglioma, breast cancer, carcinoid tumor, carcinoma, cervical cancer,colon cancer, endometrial cancer, esophageal cancer, extrahepatic bileduct cancer, ewings family of tumors (pnet), extracranial germ celltumor, eye cancer, intraocular melanoma, gallbladder cancer, gastriccancer, germ cell tumor, extragonadal, gestational trophoblastic tumor,head and neck cancer, hypopharyngeal cancer, islet cell carcinoma,laryngeal cancer, leukemia, acute lymphoblastic, leukemia, oral cavitycancer, liver cancer, lung cancer, small cell, lymphoma, AIDS-related,lymphoma, central nervous system (primary), lymphoma, cutaneous T-cell,lymphoma, hodgkin's disease, non-hodgkin's disease, malignantmesothelioma, melanoma, merkel cell carcinoma, metasatic squamouscarcinoma, multiple myeloma, plasma cell neoplasms, mycosis fungoides,myelodysplastic syndrome, myeloproliferative disorders, nasopharyngealcancer, neuroblastoma, oropharyngeal cancer, osteosarcoma, ovarianepithelial cancer, ovarian germ cell tumor, ovarian low malignantpotential tumor, pancreatic cancer, exocrine, pancreatic cancer, isletcell carcinoma, paranasal sinus and nasal cavity cancer, parathyroidcancer, penile cancer, pheochromocytoma cancer, pituitary cancer, plasmacell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renalcell cancer, salivary gland cancer, sezary syndrome, skin cancer,cutaneous T-cell lymphoma, skin cancer, kaposi's sarcoma, skin cancer,melanoma, small intestine cancer, soft tissue sarcoma, soft tissuesarcoma, testicular cancer, thymoma, malignant, thyroid cancer, urethralcancer, uterine cancer, sarcoma, unusual cancer of childhood, vaginalcancer, vulvar cancer, or wilms' tumor.

In some embodiments, the cancer is a lung cancer.

In some embodiments, the cancer is an ovarian cancer.

In some embodiments, the cancer is a triple negative breast cancer.

In some embodiments, cancer is a non-solid tumor such as a blood cancer.In another embodiment, a non-solid tumor or blood cancer is leukemia orlymphoma. In another embodiment, a non-solid tumor or blood cancer isacute lymphoblastic leukemia (ALL). In another embodiment, a non-solidtumor or blood cancer is acute myelogenous leukemia (AML). In anotherembodiment, a non-solid tumor or blood cancer is chronic lymphocyticleukemia (CLL). In another embodiment, a non-solid tumor or blood canceris small lymphocytic lymphoma (SLL). In another embodiment, a non-solidtumor or blood cancer is chronic myelogenous leukemia (CML). In anotherembodiment, a non-solid tumor or blood cancer is acute monocyticleukemia (AMOL). In another embodiment, a non-solid tumor or bloodcancer is Hodgkin's lymphomas (any of the four subtypes). In anotherembodiment, a non-solid tumor or blood cancer is Non-Hodgkin's lymphomas(any of the subtypes). In another embodiment, a non-solid tumor or bloodcancer is myeloid leukemia.

For use in the methods of the invention, the reactivating peptides maybe formulated in a conventional manner using one or morepharmaceutically acceptable carriers, stabilizers or excipients(vehicles) to form a pharmaceutical composition as is known in the art,in particular with respect to protein active agents. Carrier(s) are“acceptable” in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipient thereof.Suitable carriers typically include physiological saline or ethanolpolyols such as glycerol or propylene glycol.

The reactivating peptides may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups) and which are formed with inorganicacids such as hydrochloric or phosphoric acids, or such organic acidssuch as acetic, oxalic, tartaric and maleic. Salts formed with the freecarboxyl groups may also be derived from inorganic bases such as sodium,potassium, ammonium, calcium, or ferric hydroxides, and organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine andprocaine.

The compositions may be suitably formulated for intravenous,intramuscular, subcutaneous, or intraperitoneal administration andconveniently comprise sterile aqueous solutions of the reactivatingpeptides, which are preferably isotonic with the blood of the recipient.Such formulations are typically prepared by dissolving solid activeingredient in water containing physiologically compatible substancessuch as sodium chloride, glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be prepared in unit ormulti-dose containers, for example, sealed ampoules or vials.

The compositions may incorporate a stabilizer, such as for examplepolyethylene glycol, proteins, saccharides (for example trehalose),amino acids, inorganic acids and admixtures thereof. Stabilizers areused in aqueous solutions at the appropriate concentration and pH. ThepH of the aqueous solution is adjusted to be within the range of5.0-9.0, preferably within the range of 6-8. In formulating thereactivating peptides, anti-adsorption agent may be used. Other suitableexcipients may typically include an antioxidant such as ascorbic acid.

The compositions may be formulated as controlled release preparationswhich may be achieved through the use of polymer to complex or absorbthe proteins. Appropriate polymers for controlled release formulationsinclude for example polyester, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, and methylcellulose. Another possible method forcontrolled release is to incorporate the reactivating peptides intoparticles of a polymeric material such as polyesters, polyamino acids,hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

In some embodiments, the reactivating peptides of the invention may beformulated in peroral or oral compositions and in some embodiments,comprise liquid solutions, emulsions, suspensions, and the like. In someembodiments, pharmaceutically-acceptable carriers suitable forpreparation of such compositions are well known in the art. In someembodiments, liquid oral compositions comprise from about 0.001% toabout 0.9% of reactivating peptides, or in another embodiment, fromabout 0.01% to about 10%.

In some embodiments, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in some embodiments areaqueous solutions or emulsions comprising a safe and effective amount ofa reactivating peptide and optionally, other compounds, intended fortopical intranasal administration.

In some embodiments, injectable solutions of the invention areformulated in aqueous solutions. In one embodiment, injectable solutionsof the invention are formulated in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. In some embodiments, for transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In some embodiments, formulations for injection are presentedin unit dosage form, e.g., in ampoules or in multidose containers withoptionally, an added preservative. In some embodiments, compositions aresuspensions, solutions or emulsions in oily or aqueous vehicles, andcontain formulatory agents such as suspending, stabilizing and/ordispersing agents.

The reactivating peptides of the invention may be administered by anysuitable administration route, selected from oral, topical, transdermalor parenteral administration.

According to some embodiments the route of administration is via topicalapplication selected from dermal, vaginal, rectal, inhalation,intranasal, ocular, auricular and buccal.

According to some embodiments the route of administration is viaparenteral injection. In various embodiments, the step of administeringis carried out by a parenteral route selected from the group consistingof intravenous, intramuscular, subcutaneous, intradermal,intraperitoneal, intraarterial, intracerebral, intracerebroventricular,intraosseus and intrathecal. For example, the reactivating peptides maybe administered systemically, for example, by parenteral routes, suchas, intraperitoneal (i.p.), intravenous (i.v.), subcutaneous, orintramuscular routes. The reactivating peptides of the invention and/orany optional additional agent may be administered systemically, forexample, by intranasal administration. The reactivating peptides of theinvention and/or any optional additional agent may be administeredsystemically, for example, by oral administration, by using specificcompositions or formulations capable of providing oral bioavailabilityto proteins. The reactivating peptides of the invention and/or anyoptional additional agent may be administered locally.

The reactivating peptides may be administered in the range of about 0.1to about 20 mg/kg of subject weight, commonly about 0.5 to about 10mg/kg, and often about 1 to about 5 mg/kg. In some cases it may beadvantageous to administer a large loading dose followed by periodic(e.g., weekly) maintenance doses over the treatment period. Thereactivating peptides can also be delivered by slow-release deliverysystems, pumps, and other known delivery systems for continuousinfusion. Dosing regimens may be varied to provide the desiredcirculating levels of particular reactivating peptides based on itspharmacokinetics. Thus, doses are calculated so that the desiredcirculating level of therapeutic agent is maintained.

Typically, the effective dose is determined by the activity of thereactivating peptides and the condition of the subject, as well as thebody weight or surface area of the subject to be treated. The size ofthe dose and the dosing regime is also determined by the existence,nature, and extent of any adverse side effects that accompany theadministration of the reactivating peptides in the particular subject.

In some embodiments, there is provided a kit for treating or preventinga p53 related condition. In some embodiments, the kit comprises acontainer (such as a vial) comprising a Mut-p53 reactivating peptide ina suitable buffer and instructions for use for administration of thereactivating peptide.

It is suggested that the efficacy of treatment with the peptides of theinvention may be augmented when combined with gold standard treatments(e.g., anti-cancer therapy). Thus, the peptide can be used to treatdiseases or conditions associated with p53 (as described hereinabove)alone or in combination with other established or experimentaltherapeutic regimen for such disorders. It will be appreciated thattreatment with additional therapeutic methods or compositions has thepotential to significantly reduce the effective clinical doses of suchtreatments, thereby reducing the often devastating negative side effectsand high cost of the treatment.

Therapeutic regimen for treatment of cancer suitable for combinationwith the peptides of some embodiments of the invention or polynucleotideencoding same include, but are not limited to chemotherapy,radiotherapy, phototherapy and photodynamic therapy, surgery,nutritional therapy, ablative therapy, combined radiotherapy andchemotherapy, brachiotherapy, proton beam therapy, immunotherapy,cellular therapy and photon beam radiosurgical therapy. According to aspecific embodiment, the chometherapy is platinum-based.

Anti-Cancer Drugs

Anti-cancer drugs that can be co-administered with the compounds of theinvention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-1b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride. Additional antineoplastic agents include those disclosedin Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A.Chabner), and the introduction thereto, 1202-1263, of Goodman andGilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition,1990, McGraw-Hill, Inc. (Health Professions Division).

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Materials and Methods

Purification of Recombinant Full Length (FL) Proteins from Sf9 Cells:Mutant p53 R249S, Mutant p53 R175H and WT p53:

2×10⁷ sf9 cells in the log-phase were grown in nine 175 cm² flaskscontaining 25 ml of media and incubated overnight at 27° C.Baculoviruses containing a recombinant p53 were added into each flask,and incubated for 72 hrs. Cells were scraped from the flasks, andcentrifuged at 4° C. (3200 g for 5 mM), the media was removed and thecell pellet was washed twice with ice-cold isotonic buffer (10 mMNa₂HPO₄, pH 7.2, 130 mM NaCl, 1 mM DTPA—diethylenetriaminepentaaceticacid). To lyse cells, the cells were resuspended in 50 ml of Buffer A(20 mM Tris-HCl, pH 8.0, 12% sucrose, 2 mM EGTA, 2 mM PMSF, 5 mM DTT)with 0.2% Triton X-100 by gentle inversion. Nuclei centrifuged at 5600Gfor 8 mM and the supernatant was removed. Nuclei was lysed by adding 20ml of Buffer B (20 mM Tris-HCl, pH 8.0, 12% sucrose, 2 mM EGTA, 2 mMPMSF, 10 mM DTT+protease inhibitors) with 0.5M NaCl and were vortexedvigorously and incubated for 20 mM on ice. The nuclear lysate wastransferred to ultracentrifuge tubes and centrifuged at 100,000 g for 60min at 4° C. The supernatant was removed and diluted with Buffer B to afinal concentration 0.04 M of NaCl, then centrifuged at 20,000 g for 5mM at 4° C. The nuclear lysate was loaded onto a 5 ml Hitrap Q FF (fastflow) (Amersham Pharmacia) ion-exchange column, pre-washed with 50 ml ofbuffer A. Then, the column was washed with buffers containing highersalt concentrations to elute the protein. For example, in the case ofthe mutant p53 R249S, the protein eluted from the Ion exchange column at˜150 mM NaCl. The protein was further purified by gel-filtrationchromatography using a preparative Superdex 75 column (AmershamPharmacia Biotech), pre-equilibrated with 20 mM sodium citrate pH 6.1,150 mM NaCl, 10 μM ZnCl₂, and 10 mM DTT. Fractions containing purifiedprotein were pooled together and concentrated to 6-7 mg/ml, aliquotedand stored at −80° C. The fractions obtained after each purificationstep were analyzed on dot-blot for presence of mutant p53 andsubsequently on SDS-PAGE with Coomassie blue staining to check purity ofthe fractions.

Sandwich ELISA

96-well plates were coated using 3 different antibodies (1 type ofantibody (Ab) in each well): PAb421 recognizes both conformations of p53and binds to a C-terminus epitope; PAb240 recognizes mutant conformationof p53, binds to epitope within the core domain (amino acids 212-217)(Stephen, C. W. and D. P. Lane, Mutant conformation of p53. Preciseepitope mapping using a filamentous phage epitope library. J. Mol.Biol., 1992. 225(3): p. 577-83) which is accessible to the Ab when theprotein is partially denatured (for example, when the DBD is mutated);and PAb1620, which recognizes WT conformation of p53, binds to epitopewith in the core domain (aa 156, 206-210), formed when folding is in WTconformation (Wang, P. L., F. Sait, and G. Winter, The ‘wild type’conformation of p53: epitope mapping using hybrid proteins. Oncogene,2001. 20(18): p. 2318-24).

Wells were incubated overnight (ON) with 100 μl Ab (5 μg/ml) in roomtemp (RT). The liquid was discarded, and the wells were washed 3 timeswith Phosphate buffered saline (PBS), 200 μl per each wash. Next,blocking with 200 μl of 5% bovine serum albumin (BSA) diluted in PBS ineach well for 1.5 hours at room temperature (RT) was performed. Blockingbuffer was discarded, followed by 3 washes in PBS as described above.Samples of mutant and WT p53 proteins (100 μl, 10 μg/ml), together withcontrol peptides pCAP-710 (LPNPPER, SEQ ID NO:340) and pCAP-1220(FRSFAIPLVVPF, SEQ ID NO:368) (5 μg/ml, Sigma Aldrich, or with testpeptides 1-153 (5 μg/ml), were incubated for 1.5 hours together, andthen added to the wells. Samples were rotated and incubated for 1 hourat RT. Samples were discarded, following 4 washes as described above,using Trisphosphate buffered saline (TPBS). Next, horseradish peroxidase(HRP) conjugated streptavidin p53 antibody (10 μg/ml HAF1355 (R&D)) wasadded to the wells and incubated at RT for 1 hour. After the plate waswashed 3 times in TPBS, TMB substrate solution (50 μl each well, Thermo,(Cat. No. ES001-1L-K)) was added and incubated at 37° c. for 20 min. Thereaction was stopped with 2M sulfuric acid (50 μl). The absorbance wasmeasured at 450 nm with a spectrophotometer. Protein concentration wasdetermined by dividing the absorbencies of each sample to the absorbanceof Ab 421 samples.

DNA Binding Assay

For these experiments, a commercial p53/DNA binding kit of “R&D”(Cat-DYC1355-5 Lot-1273366FA) was used, in accordance with manufacturerguide lines. Briefly, 96 well plates are coated with anti-p53 antibodyovernight. Cell extracts containing p53 are reacted with anoligonucleotide that contains a p53 consensus binding site (provided inthe kit), labeled with biotin, in the presence or absenc.

e (NT) of test peptides. WT p53 is expected to bind this DNA bindingsite as well as to the antibody coating the test wells of the plate.Excess p53 and oligos were washed away and streptavidin-HRP was used toquantify the amount of oligos in the well, which is proportional to theDNA bound by p53. TMB assay was performed to determine HRP (ES001-1L-K)levels (450 nm).

Crystal Violet Assay

Cells were cultured in 96 wells plates with 2500-4000 cells/well in 0.1ml and incubated overnight at 37° C. in order to adhere to the plate.Serial dilutions of different peptides (0.5 μg/ml) were added in 0.1 mlaliquots and the plates incubated for additional 48 h at 37° C. Thenmedium was removed and cell lysis was determined by staining the cellswith crystal violet (0.5%) in methanol/water (1:4, v/v), 50 μl eachwell, for 10 min, followed by 3 washes with PBS. Afterwards, 10% aceticacid (50 μl) was added to each well and shaken for 10 min. Then,automatic plate reading was performed at 595 nm.

Immunofluorescence

Cells were cultured on cover slips overnight and then were treated withpeptides using X-fect transfection. After 2 hour recovery, cells werefixed with 4% paraformaldehyde for 30 min at room temperature followedby 3 washes (PBS). Samples were permeabilized with 0.1% Triton (1% BSAin PBS) for 10 min RT followed by blocking (3 washes of 0.5% BSA inPBS), 5 min each wash. Cells were then probed with a mouse anti-p53(DO-1) antibody diluted 1:500 for 1.5 hours, followed by blocking (3washes of 0.5% BSA in PBS), 5 min each wash. Then cells were probed withgoat anti-mouse Cy3 diluted 1:600 and DAPI diluted 1:1000 for 45 minSamples were mounted with Elvanol.

Luciferase Assay Construction of Luciferase Constructs

The oligonucleotide (RGC-W) that has the sequence5′-TCGAGTTGCCTGGACTTGCCTGGCCTTGCCTTTTC-3′ (SEQ ID NO:362), and theoligonucleotide mutant RGC oligonucleotide (RGC-M) that has the sequence5′-TCGAGTTTAATGGACTTTAATGGCCTTTAATTTTC-3′ (SEQ ID NO:363), are bothderived from Kern et al. (Kern, S. E., et al., Identification of p53 asa sequence-specific DNA-binding protein. Science, 1991. 252(5013): p.1708-11), and serve as a consensus binding sites for WT p53.

These motifs were cloned into the KPN and Eco53IK sites in pCLuc Mini-TK2 Vector (NEB, Cat No. N03245). The Luciferase construct was used toassess transcriptional activation of p53 in test cells.

ChIP Analysis

Briefly, clones were cross-linked with formaldehyde (1% finalconcentration) at room temperature for 10 min. The formaldehyde wasneutralized with 2.5M glycine (final concentration 0.25M) for 5 mM Cellswere washed sequentially with 1 ml of ice-cold PBS, buffer I (0.25%Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5), and bufferII (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5) andharvested by scraping. Cells were then resuspended in 0.3 ml of lysisbuffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1× proteaseinhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, Ind.)and sonicated 10 times (20 sec ‘on’ followed by 40 sec ‘off’) at themaximum setting (Biorupter, Diagenode, NY) followed by centrifugationfor 10 min on ice to produce 200-500 bp fragments. Supernatants werecollected and diluted 10 times in the ChIP dilution buffer (1% TritonX-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) followed byimmuno-clearing with 40 μl of pre-blocked protein A-sepharose (SantaCruz Biotech) with 2 μg sheared salmon sperm DNA and pre-immune serum (1μg of rabbit serum with 10 μl of 100 mg/mL BSA for 2 hour at 4° C. Asample was retained for the preparation of the input sample.

Immuno-precipitation was performed overnight at 4° C. with specificantibodies obtained from. After immuno-precipitation, 40 μl proteinA-Sepharose (pre-blocked with salmon sperm DNA) were added and furtherincubated for another 1 hr. Precipitates were washed sequentially for 10min each in TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl,pH 8.1, 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris-HCl, pH 8.1, 500 mM NaCl), and buffer III (0.25 M LiCl, 1%NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Precipitateswere then washed three times with TE buffer and extracted twice with 1%SDS, 0.1 M NaHCO₃. Eluates were pooled and heated at 65° C. for aminimum of 6 hour to overnight to reverse the formaldehydecross-linking. DNA fragments were purified with a QIAquick Spin Kit(Qiagen, CA). Immuno-precipitation reactions were performed intriplicate using beads only as a non-specific control. Quantitativeanalysis of the active and repressive histone marks in the ChIP productsfrom clones were assessed by quantitative real-time PCR. In order tonormalize the efficiency of immunoprecipitation (IP), the normalizationof chromatin IP was done using specific primers for necdin promoterregion and 5′ region (which corresponds to a repressive chromatinregion).

Cell Culture and Luciferase Reporter Assays

H1299 p53-null cells were cultured overnight and then transfected withthe luciferase constructs using MaxFect Transfection Agent (Mediatech)according to the manufacturer's protocol. Prior to the transfection,cell medium was exchanged to OPTI-MEM.

The cells were treated with different peptides 24 hours aftertransfection. After additional 24 hours, growth medium was collected to96 black plates: 40 μl for Cluc assay, and 20 μl for Gluc assay. Assaywas performed using Turner BioSystems Modulus Microplate. Value wascalculated by Cluc/gluc/NT (non-treated cells).

RT-PCR

RNA was obtained using Macherey-Nagel NucleoSpin RNA II Kit on cellspellet according to the manufacturer's protocol. Aliquots of 0.4-1 μgwere reverse transcribed using Bio-RT 2000 (Bio-Lab) and random hexamerprimers. Quantitative real-time polymerase chain reaction (QRT-PCR) wasperformed on an ABI 7300 instrument (Applied Biosystems) using PerfeCTaSYBR Green FastMix ROX (Quanta). RT-PCR primers used are presented inTable 1 (primers sequences are presented 5′ to 3′).

Phage Display Library

Phage display library used were commercially available phage libraries,generated by New England Biolabs (NEB). One library is of linearhepta-peptides (PhD-7), the other library is of linear dodeca-peptides(PhD-12) (CAT NOs.: PhD-7, E8100S; PhD-12, E8110S). The randomizedpeptide sequences in both libraries are expressed at the N-terminus ofthe minor coat protein pIII, resulting in a valency of 5 copies of thedisplayed peptide per virion. All of the libraries contain a shortlinker sequence between the displayed peptide and pIII.

Deep Sequencing

Prior to sequencing, a PCR reaction was performed with primers flankingthe inserted libraries Forward-5′-NNNNNNNNCATGGAAAGATAGTG (SEQ IDNO:364) and Reverse-5′-NNNNNNNNCCTAAAACGATTTGTG (SEQ ID NO:365), first 8bases of each primer are randomized and were incorporated as a mixtureof all four bases. Randomization of first bases was introduced since theSolexa sequence equipment is incapable of sequencing repetitivesequences for the first few cycles. The PCR reaction yielded DNA in therequired quantity 5 ug and length (about 120 bp) which includes theflanking primers and the cloned peptide library for Solexa deepsequencing.

Example 1 Calibration of Experimental Conditions Choosing a p53 ProteinSource

When choosing the protein source for phage display selection, severalconsiderations are taken into account; the use of purified proteins isrecommended since interaction of phage clones with different proteins insolution can give rise to nonspecific false positive peptides. The humanfull length p53 protein purified from SF9 cells (see above), was used inthe following experiments (Accession No. CG3336). Therefore, anexpression system of p53 in SF9 insect cell line infected by baculovirus(as detailed above) was used. A major advantage of p53 expressed in thissystem is that it already contains post translational modifications.

Conformation of Baculovirus-Expressed WT p53 and Mut-p53 Proteins

Initial experiments with the Baculo-p53 were made by using the nuclearextracts lysates of Sf9 cells expressing either WT p53, a hot spot fulllength mutant p53 (R175H), or temperature sensitive (ts) mutant p53(V143A). SF9 cells were infected with viruses caring either one of thethree expressing vectors. 48 hours after infection cells were harvested,nuclei extracted and the extracts were subjected to immunoprecipitationwith: PAb1620, PAb240, ASPP2 (also named (P53-BP2)) and/or Bcl2 for 3hours at 4° C. The immunoprecipitated p53 was detected by westernblotting using the αp53-HRP Ab (Cat No. HAF1355 (R&D)). The results ofthis IP-Western experiment are shown in FIG. 2. As can be seen, thetemperature sensitive (ts)-mutant p53 V143A (4° C.) and the WT p53 bothbind well to the PAb1620 antibody, but not to PAb240. On the other hand,the mutant p53 R175H exhibits stronger binding to PAb240 than toPAb1620. This suggests that Baculo-expressed mutant p53 R175H assumes aconformation that is an intermediate between mutant and wild type p53.Bcl2 does not exhibit binding to either one of the p53 forms, whereasASPP2 (P53-BP2) binds to all forms of p53 with approximately the sameaffinity. Therefore, it is concluded that ASPP2 and Bcl2 cannot be usedas markers of p53 conformation under these experimental conditions.

Calibration of Solution Conditions

In order to reduce the relatively high residual binding of the mutantp53 R175H to the PAb1620 and to enhance the binding of WT p53 to thatantibody, fine tuning of assay conditions was performed. The results areshown in FIG. 3 which shows a blot of the purified mutant p53 (R175H)and WT p53, extracted from nuclei of Sf9 cells infected with thecorresponding baculovirus (as described above). The purified p53 wasdissolved in the specified buffers (A-Tris-50 mM; B-Tris, NaCl 150 mM;C-Tris, NaCl, Triton 0.5%; D-Tris, Glicyn 0.5%; E-Na4O7P2 40-mM; F-GndCl400 mM; G-GndCl 800 mM; H-Urea 1M; I-Urea 3M; IP-IP Buffer) and thenimmunoprecipitated with PAb1620 and PAb240 for 3 hours at 4° C. andsubjected to western blotting using the αp53-HRP-Ab. As can be seen,solution (A) contains only 50 mM Tris. In this solution the binding ofmutant p53 R175H to PAb1620 is only about 5% compared to that bound toPAb240. Addition of either 150 mM NaCl (B), 150 mM NaCl+0.5% Triton (C)or 0.5% glycine (D) enhanced the binding of mutant R175H to PAb1620. 3MUrea (I) reduced the binding of p53 mutant R175H to PAb1620, probably bycausing denaturation of the protein. A lower concentration of urea, 1M(H), increased the binding of mutant p53 R175H (R175H p53) to PAb1620.40 mM Na₄O₇P₂ (E) reduced the binding of R175H p53 to PAb1620 to thelowest level. Finally, in IP buffer the, R175H p53 remained PAb1620negative; however in this buffer WT p53 showed strong PAb 240 bindingand reduced binding to PAb1620, suggesting that IP buffer causes mildmisfolding of the WT form. Hence, buffer containing Tris only is usedfor further experiments.

Example 2 Initial Screening of Phage Display Library and Selecting forMut-p53 Reactivating Peptides

A phage display screen, using the R175H p53 protein, a single phd-12phage library (NEB, Cat. No. E8110S) and selection with PAb1620 antibodywas initially performed. 200 ng of R175H p53 were reacted with 10¹¹phage for 1 hour to allow binding of presented peptides of the phage tothe Mut-p53 (R175H). Next, beads cross linked to PAb1620 were added foran additional 1 hour to immunoprecipitate the entire complex. Thispanning procedure was repeated for three rounds, increasing thestringency of the selection after each round by reducing the amount ofincubated Mut-p53: 1^(st) round 200 ng, 2^(nd) round 100 ng and 3^(rd)round 50 ng. Phages were eluted using purified WT p53 DBD, at aconcentration of 2 μg/ml (p53 DBD (residues 94-293) was sub-cloned intopET-27b (Novagen)). The plasmid was transformed into E. coli BL21 (DE3)strain. Protein production was conducted following a procedure describedfor the mouse p53 DBD (Suad, O., et al., Structural basis of restoringsequence-specific DNA binding and transactivation to mutant p53 bysuppressor mutations. J Mol. Biol., 2009. 385(1): p. 249-65). After eachround of selection, tittering of the eluted phage was performed, to getan estimate of the number of phages that were selected (Table 2). Theeluted phages were amplified by infecting E-coli, to yield about 10¹³phage for selection in the next round. From the second round of panning,a control panning experiment was performed with PAb1620 only (withoutincubation with Mut-p53); this titer is indicative of the specificity ofthe panning.

As seen in Table 2, 100 infectious phage particles/pi were obtained inthe first selection round and typical enrichment values betweenselection rounds, giving rise to higher enrichment in the first coupleof rounds and then reaching a plateau in the third and fourth roundpanning. However, the number of phage eluted in both the specificselection panning reactions as well as in the nonspecific PAb1620control panning reactions was similar. Such enrichment suggests that thephage may bind directly to the PAb1620 and not through interaction withthe p53 R175H target.

In order to reduce background (nonspecific binding), additionalpre-clearing steps and increasing pre-clearing time were introduced;however, the proportion of background binding remained high. Therefore,alternating selection steps during the phage display process wereimplemented, in order to reduce background binding. To this aim,different selection strategies at each selection round, while trying tominimize common nonspecific elements in the experimental system (andhence reducing binding to those nonspecific elements) were performed.

Since it is assumed that a prerequisite of conformational change of p53is the binding of a peptide to p53, an additional selection step for WTp53 binding in between the PAb1620 selections was introduced. It washypothesized that since PAb1620 would not be present in the secondpanning round, the phage binding directly to it would be eliminated.Moreover, since a prerequisite of any functional peptide is binding top53, peptides preferentially binding to the WT form are expected tostabilize this conformation. The first and third rounds of panning weresimilar to the previous experiment. In the second selection round,however, a selection for phage binding for WT-p53 (His tagged) wasperformed, and the p53/phage complex was immunoprecipitated using nickelbeads (which bind to the His tag). The titer of the eluted phage wasevaluated after each selection round. As shown in Table 3, 10-foldenrichment was achieved in the elution of phage when the second cyclewas compared to the first. Although this may be considered a bit low byphage display standards, the reason for this relatively low enrichmentis probably the use of different selection strategies in each round ofpanning, increasing the specificity but on the other hand reducing theoverall yield of selected phage. The enrichment from the secondselection round to the third was in the order of 100 fold, indicating amarked increase in phage enrichment, compared to the previous factor of10. This marked increase is due to the repeated PAb1620 selectionImportantly, the number of phages after the third round was in the orderof 10⁵, whereas with the control PAb1620 it was 4×10³. Therefore, thenonspecific control (i.e., background), constitutes only about 5% of thetotal selected phage.

Example 3 Method for Screening, and Identifying Mut-p53 ReactivatingPeptides

In order to screen, identify and isolate specific p53 reactivatingpeptides, a method which uses a combination of different andcomplementary selection strategies was devised and performed.

In this example, three selection strategies were combined. The firstselection strategy relies on the reactivity with PAb1620, as describedabove. The second selection strategy is based on the binding of WT p53to its consensus DNA sequence motif: p53 responsive element (p53-RE).The binding of p53 to its consensus DNA in-vitro has been extensivelydemonstrated Poerger, A. C., M. D. Allen, and A. R. Fersht, Crystalstructure of a superstable mutant of human p53 core domain. Insightsinto the mechanism of rescuing oncogenic mutations. J Biol Chem, 2004.279(2): p. 1291-6). Accordingly, two complementary oligonucleotides weredesigned to produce dsDNA (after annealing). These oligonucleotidescontain two tandem copies of a p53-RE consensus sequences: one consensussequence is the perfect consensus binding site, deduced from bindingexperiments (AGACATGCCCAGACATGTCC (SEQ ID NO:339)) and the othersequence is a p53 DNA binding site, derived from the p21 promoter(GAACATGTCCCAACATGTTG (SEQ ID NO:340)), which is located downstream tothe first consensus sequence (FIG. 4). In addition, two restrictionenzyme sites (HindIII (AAGCTT (SEQ ID NO:341)) and EcoRI (GAATTC (SEQ IDNO:342)), which enable a more specific elution step after selection werefurther introduced. One oligonucleotide strand was also labeled withbiotin, to allow immunoprecipitation of DNA/p53/phage complex withstreptavidin coated beads. FIG. 4 shows a schematic sequence of thep53-RE oligonucleotide and the sequence elements thereof. The sequenceof the upper strand oligonucleotide is:

Biotin-5'- (SEQ ID NO: 361) CTGCTGAAGCTTCGAATTCCT

TACTGC TGCTGCTGCTGCTGCTGC

CTGCTGCTG CTGCTG-3'.

In a selection procedure performed using the DNA binding strategy (asdetailed below), 0.5-3 pmol of the biotin-p53-RE oligonucleotide wasreacted with 200 ng of purified WT p53 for 1 hour to allow binding. 10¹⁰phage from either PhD-7 or PhD-12 phage libraries were then introducedfor an additional hour. Next, streptavidin coated agarose beads wereadded for 30 minutes. 5-12 washing steps were then performed, followedby elution performed by adding either the restriction enzymes or anexcess of non-biotinylated DNA for 30 minutes. These precautions wouldreduce selection of phage binding to DNA, biotin and streptavidin.

The third selection strategy is based on the SV40 large T (LT) antigen.The binding between p53 and SV40 LT is considered to be very strong.Therefore, p53 has to be folded properly to form the binding epitopeplatform to SV40 LT. To this aim, Sf9 cells were infected withbaculovirus encoding for SV40 LT. Cells were lysed and the SV40 LT wasisolated using protein-A beads cross-linked to PAb 419 (antibodyspecific for SV40 LT, (Abcam-ab1684)). Beads were washed several times,and then used for phage display selections. The panning procedure forSV40 LT binding was similar to the conformation based strategy, exceptthat instead of using PAb1620 beads, PAb 419-SV40 LT beads were used forthe selection.

A combination of all three selection strategies in alternating roundsyields the best results, since each cycle gradually increases thepercentage of phage that harbor the desired specific peptides, whilereducing non-specific background. A schematic illustration of the methodof identification and selection is illustrated in FIGS. 1A and 1B.

Phage display screening was performed in parallel with PhD-7 and PhD-12phage peptide libraries. Alternating cycles of phage selection, using adifferent immobilized platform (PAb1620, p53-RE DNA or SV40 LT) at eachstep were performed. Table 4 shows the different selection routes takento produce enriched phage libraries, and specifies the titer valuesafter each round of selection. By using such different combinations ofselection platforms (e.g. PAb1620 followed by p53 consensus DNA followedagain by PAb1620, or SV40 LT followed by PAb1620 followed by SV40 LT),as well as the 2 different phage libraries, a panel of sub-libraries wasobtained, that could then be compared after sequencing. After 3 cyclesof selection, over 60 different pools (sub-libraries) containing a highproportion of Mut-p53-reactivating phage (Table 4) were obtained.

Example 5 Selected Phage Pools Induce Binding of Mut-p53 to PAb1620

To determine whether the phage display selection method as performedabove can enrich for phage that reactivate Mut-p53, the ability of thephage pools obtained after 3 cycles of selection to induce the bindingof either full length R175H Mut-p53 (BD Pharmingen, Cat. No. 556439), orthe recombinant R249S p53 DBD (249 DBD) proteins to PAb1620 was tested.To reduce the undesirable effect of contaminating phage that exhibitdirect binding to PAb 1620, a pre-clearing step was included whereby thephage pool was first incubated with PAb1620 only, before being added tothe test reaction. Beads covalently cross-linked to PAb1620 wereincubated with purified mutant p53 R175H in the presence of phageobtained by phage display selection with either Mut-p53 R175H (175) orMut-p53 R249S (249), either without or with prior pre-clearing stepperformed by incubation of the phage pool with PAb1620 beads. Nonselected phage (ns) were used as control. Incubation was performed for 3hours at 4° C. Bound p53 was visualized by western blot analysis usingantibody against p53. As can be seen in the results presented in FIG. 5,some of the selected phage pools indeed induced binding of Mut-p53 toPAb1620, as compared to no phage or non-selected input phage (ns).

Example 6 Selected Phage Pools Induce Binding of Mut-p53 to p53Consensus DNA

To further test whether the selected phage pools can facilitate thebinding of Mut-p53 to p53 consensus DNA binding element, biotin-labelledoligonucleotides corresponding to the p53 responsive element consensus(p53-RE) biotin-AGACATGCCCAGACATGTC CTTATAGACATGCCCAGACATGTCC (SEQ IDNO:366) or control oligonucleotides mutated in key residues crucial forp53 binding (Con-RE biotin-AGAaATGCCCAGA aATGTCCTTATAGAaATGCCCAGAaATGTCC(SEQ ID NO:367), were immobilized by reacting these oligos withstreptavidin coated beads. The p53-RE or Con-RE beads were incubatedwith either WT p53 DBD or mutant 249 DBD, together with the phage poolsobtained after 3 cycles of selection. Streptavidin coated beads boundeither to p53-RE-DNA or Con-RE-DNA oligonucleotides, labelled withbiotin, were incubated with purified WT p53-DBD or mutant p53 R249S-DBDin the presence of phage obtained by phage display selection withMut-p53 R175H (175), clone 27 (LPNPPER, SEQ ID NO:328) (a single cloneisolated from the R175H selection), pools #69 and #94, selected with WTand Mut-p53 R175H using combinations of T-AG and PAb1620 at alternatingselection rounds. Non selected phage (NS) were used as control.Incubation was for 3 hours at 4° C. Bound p53 was visualized by westernblot analysis. As can be seen in the results presented in FIG. 6, the WTp53 DBD bound to p53-RE better than to the Con-RE, as expected. The249DBD did not bind to the p53-RE, consistent with its known loss ofsequence-specific DNA binding ability. Importantly, the selected phagepools were capable of inducing the binding of Mut-p53 to the p53-RE,demonstrating that they are indeed capable of reactivating and restoringthe lost function of Mut-p53.

Example 7 Deep Sequencing of Selected Phage Pools

Next generation sequencing, which greatly increases the effectiveness ofphage display, allowing extraction and analysis of the entire selectedpeptide repertoire, with fewer selection cycles was performed. Eightphage pools were selected for deep sequencing using criteria ofincreased enrichment between selection rounds and functional activity.Prior to sequencing, a PCR reaction was performed with primers flankingthe inserted libraries: Forward-5′-NNNNNNNNCATGGAAAGATAGTG (SEQ IDNO:364), and Reverse-5′-NNNNNNNNCCTAAAACGATTTGTG (SEQ ID NO:365), thefirst 8 bases of each primer are randomized and were incorporated as amixture of all four bases. Randomization of first bases was introducedto improve sequencing efficiency and accuracy. The PCR reaction yieldedDNA in the required quantity 5 μg and length (about 120 bp), whichincludes the flanking primers and the cloned peptide library for Solexadeep sequencing.

The deep sequencing yielded a database of 36 million reads. 95% of thesequences contained the primer sequences used in the PCR when extractingthe libraries. Next, a preliminary bioinformatics analysis of the datawas performed. This analysis included the removal of sequences that donot contain the original primers, removal of sequences that are not inthe correct reading frame, segregation of the database into the original12 amino-acid and 7 amino-acid libraries according to insert length, andfinally counting of unique sequences and sorting them according tonumber of appearances in the database. It was found that most sequencesappeared only once or twice in the database, presumably corresponding tobackground phage. 12 reads were defined as a cutoff, beneath which theenrichment of sequences was considered to be insignificant. The DNAsequences in the database were then translated into amino acidsequences.

As an internal quality control, the sequences and their abundance as thepercent from the total library between the two strands that weresequenced from opposite directions and therefore contained a differentprimer at their 5′ were compared. The comparison showed that thesequences and their abundance was similar between the two strands,indicating that the obtained sequence database is valid.

Table 5 shows a list of peptide sequences obtained from the deepsequencing database of 5′ strands. This database contains 10⁷ sequencesin total, after filtering irrelevant sequences. A cut-off counting andtranslation was then performed. The column (#Reads) shows the number oftimes the sequence repeats in the described database and thereforecorresponds to the enrichment of that specific sequence. Since thebioinformatics analysis was performed on DNA sequences, and individualpeptides can be encoded by several different DNA sequences because ofthe genetic code degeneracy, there are quite a few peptides that appearin the table more than once. If a certain peptide is encoded bydifferent DNA sequences, it means that it was selected independentlywithin different phage clones.

Alternatively, a number of DNA sequences coding for the same peptidecould be a result of sequencing errors: however, in this case it wouldbe expected that the result of such a mistake would be in a random baseand therefore not enriched in a high number of reads. Therefore, DNAsequences that were under 30 reads in the #Repeats count were excluded.The column (#Repeats) shows the number of DNA sequences coding for thesame peptide sequence, and is therefore a further indication of thespecificity and strength of the selection.

As seen in Table 5, the sequences could be segregated into their twolibraries of origin. The peptide sequence is depicted in the middlecolumn and the sequences are sorted in descending order according to thenumber of reads that corresponds to the enrichment in each library. The12aa library was found to be dominated by a single sequence—KPPDRLWHYTQP(SEQ ID NO:322), that makes up almost 20% of the total number ofsequences. The 7aa library is more diverse and contains many moresequences, but with lower enrichment values.

Table 5 presents the analysis of deep sequencing data base—sequences aredivided into to their two libraries of origin, the peptide sequence isdepicted in the middle column and the sequences are sorted in descendingorder according to the number of reads that corresponds to theenrichment in each library. The column (#Repeats) shows the number ofDNA sequences coding for the same peptide sequence.

Example 8 Bioinformatics Motif Analysis of the Deep Sequencing Database

Next, a more comprehensive bioinformatics analysis was performed inorder to identify consensus motifs. Such motifs could be elucidated inseveral ways. First, comparison between peptide sequences identified inthe 12aa and the 7aa libraries. The appearance of common motifs in bothlibraries would support the strength of such a motif since it wasclearly selected in two completely independent experiments. Secondly,the abundance of a certain amino acid in a particular position and itssimilarity to other amino acids in the same position of the motif canserve as an indication for the significance of such amino acid in thisparticular position. Thirdly, the position of a motif may be of criticalimportance to its function: a short motif can shift along a longerpeptide sequence with variability in other amino-acid sequences and thedistance from the free N-terminus of the peptide may inform onsignificance to its activity. An algorithm was developed to check theamino acid sequence in a growing window of peptide length as follows:

-   -   1. scoring each peptide, integrating the number of different        nucleotide sequences that translate into the same peptide with        the occurrences of each such type of nucleotide sequence;    -   2. clustering the different peptides, scoring the sequence        similarity between different peptides; and    -   3. identifying groups of related peptide sequences and        extracting a consensus therefrom.

Candidate peptides were those with the top occurrences ≧0.2%: 40 fromthe 7aa library, and 32 from the 12aa library. These could be clusteredinto 40 groups by their Blastp similarities and occurrence of a shortamino acid (aa motif). Most groups included a single peptide, but 9groups included 2-13 peptides, and 6 of these groups included both 7aaand 12aa peptides.

The groups were transformed into block multiple alignments, with the %occurrences being the sequence weights. The blocks were used to querythe 7aa and 12aa peptide-clustered sequence files, and the top resultswere again transformed into blocks in the same way. In some blocks, butnot in all, results from the two libraries were similar to each other.

The deep sequencing output (i.e. creation of database of millions ofpeptide sequences as compared to hundreds of sequences by conventionalsequencing) enabled a much more detailed and comprehensive analysis ofconsensus motifs. Overall, about 130 motifs of significantly enrichedsequences were identified; most of these peptide motifs are representedby several DNA sequences and 16 of these motifs are shared between boththe 7aa and 12aa libraries. FIG. 7 shows several such motifs. Some ofthe motifs resulted from combining overlapping sequences and thereforeare longer than the original peptide libraries.

Example 9 Synthesis of Peptides

From the obtained list of peptide motifs identified as described above,128 peptides were chemically synthesized by PEPTIDE 2.0 at crude puritytaking advantage of a 96 well format. This semi high throughputsynthesis enabled a relatively low cost of each peptide. Table 6 belowlists the peptides synthesized. This list also includes some peptidesderived from proteins that are known from the literature to interactwith p53. The list also includes 10 peptides synthesized in twoversions, both without and with a poly arginine C-terminal addition.This poly-Arg addition was reported to enable the crossing of peptidesacross cell membranes. This allows the evaluation of both the ability ofthe poly Arg C-terminal addition to enable peptide delivery into thecells and whether it interferes with the activity of these particularpeptides in-vivo. The poly Arg may include 0-10 Arg residues and isdesignated as R₀₋₁₀.

Differences between the chemically synthesized peptides and the peptidesthat were selected from phage display libraries may occur. Inparticular, the selected peptides were presented in the context of thephage as fusion proteins with the pIII phage coat protein. Therefore,this transition to synthetic peptide is not trivial, and it is knownthat in some instances peptides shown to be active when presented onphage lose their activity when the same sequence is synthesized as afree peptide.

Example 10 Functional Screening of Lead Test Peptides

Several alternative and complementary methods to screen the lead peptidecandidates for conformational and functional effects on Mut-p53 wereused. Since no information regarding the penetration of each testpeptide across cell membranes was known, in-vitro based assays forevaluation were first performed: ELISA for assessment of p53conformation and sequence-specific DNA binding of p53. Subsequently, thepeptides' activity was examined in live cells by viability assays, p53transcriptional activity on a luciferase reporter gene, and examinationof p53 target genes in-vivo. Combination of these assays (all performedin a 96 well format) allowed the identification and validation of thepeptide's effects on different p53 activities and their ability toconfer such ability to Mut-p53 proteins.

Screening Peptides for Effect on p53 Conformation

The first screening strategy was based on ELISA. A version of sandwichELISA was used to examine the effect of the lead test peptides on p53conformation. To measure the conformational effect of the peptides onMut-p53, a micro-titer plate was coated with PAb240, PAb1620 or pAb421(as a positive control), and then the reactivity of p53 to theseantibodies was examined WT p53 served as a positive control forreactivity with PAb1620, and Mut-p53 served as a negative control. Toexamine the effect of a tested peptide it was added to a solutioncontaining Mut-p53 and change in reactivity to either Ab was tested. Ifafter addition of a peptide an increased reactivity of Mut-p53 towardsPAb1620 and a decreased reactivity to PAb240 were observed, it indicatedthat the tested peptide has reactivated WT conformation of Mut-p53.Several ELISA experiments using different cell extracts were performed.The results are presented in FIGS. 8A-B, which show a representativeexperiment performed on an extract of H1299 cells stably overexpressingMut-p53, (R175H p53). Extracts were prepared at 750 ng/μl concentrationin standard immunoprecipitation buffer at a physiological pH and saltconcentrations and supplemented with 3% BSA for blocking, and thenreacted with different peptides at a concentration of 50 ng/ml for 2hours. Plates were coated with the various antibodies (Abs) overnight,washed, blocked, and cell extracts (with or without peptides) were addedfor an additional 2 hours. After removal of extracts, plates were washedand incubated with the αp53-HRP conjugated Ab for the detection of p53levels. Finally, a TMB (substrate of HRP) assay was performed andoptical density was determined at 450 nm (as described above). MCF7 andH1299-Mut-p53 (ts) A135V (Zhang, W., et al., A temperature-sensitivemutant of human p53. Embo J, 1994. 13(11): p. 2535-44) cells were usedas positive controls for the WT p53 conformation (1620/240 ratio equalsor exceeds 5:1). The H1299-R175H p53 extracts, while exhibiting moremutant p53 conformation, still maintained reactivity to PAb1620(1620/240 ratio is 1:2) of PAb1620 or PAb240 over PAb421. However, whendiscussing the outcome of the analysis, the PAb1620/PAb240 calculatedratio, which better captures the extent of conformational change isreferred to. To examine whether this is background binding to theantibody or actual WT folding conformation, increasing levels ofdenaturation were induced by heating the extracts for different timelengths, monitoring their reactivity to PAb1620 and PAb240. As seen inFIGS. 8A and 8B, increased heat treatment induced an increase inreactivity with PAb240 and a decrease in reactivity with PAb1620,indicating that the R175H p53 in these extracts remained partly in WTconformation under these experimental conditions. Notably, afterincubation with some of the tested peptides, increased reactivity ofR175H Mut-p53 towards PAb1620 and decreased reactivity towards PAb240was detected. This was the case, for example, with peptides 24, 36, 47,60, 68 (Table 6), indicating that these peptides elicit a conformationalchange in mutant p53 protein.

Screening of Peptides for Effect on Mut-p53 Binding to p53-RE DNA.

To measure the effect of the tested peptides on DNA binding of Mut-p53,a commercial ELISA kit, (R&D Systems DYC1355-5, Lot-1273366FA), was usedas a high-throughput assay to quantify p53 activation. This kit uses a96-well plate format. The kit was used according to manufacturer'sinstructions. Wells were coated with anti-p53 antibody overnight. Cellextracts containing p53 were reacted with a biotin labeledoligonucleotide containing a p53 consensus binding site (included in thekit). WT p53 is expected to bind this oligo as well as the antibodycoating the wells. Excess p53 and oligo were washed away in wash buffer(0.05% Tween 20 in PBS, pH 7.2-7.4; R&D Systems, Catalog # WAl26). Then,streptavidin-HRP (R&D Systems, Part 890803, provided in the kit) wasadded for 15-45 min to quantify the amount of oligo in the well, whichis proportional to the DNA bound by p53. If the addition of a peptide toMut-p53 extracts increases ELISA reading compared to background, thispeptide is considered as functionally effective and may be selected forfurther analysis. FIG. 9 shows a representative experiment: similarly toconformation ELISA, cell extracts were incubated with biotin-p53-REeither in presence or absence (NT) of test peptides. As with theconformational screening, MCF7 and the H1299-Mut-p53 (ts) A135V cellsserved as positive controls for WT p53. Extracts were added to the wellscoated with αp53-Ab, and after several washing steps, streptavidin-HRPwas added for 1 hour, and then plates were washed again and TMB(substrate to HRP) assay was performed. As can be seen in FIG. 9,H1299-R175H p53 extracts exhibited some background binding to the p53-REoligo, which was further reduced by non-labeled competing oligo.Positive controls showed a 3-4 fold higher signal compared to thebackground. Several peptides appear to elevate the binding ofH1299-R175H p53 extracts to p53-RE DNA, for example: 68, 75, 83, 93, 97.

Binding of Peptides to WT p53 and Mutant p53.

To measure the binding of peptides to Mut-p53 and WT p53, a commercialELISA kit from “TAKARA” (MK100 Lot AK401), was used as a high-throughputassay to quantify the binding of different peptides to proteins orantibodies. The kit was used according to the manufacturer'sinstructions. The wells were plated with the peptides by performing achemical reaction attaching the C-terminus of the peptide to the plate.

Recombinant WT p53 or Mut-p53 R175H at a concentration of 10 ng/ml wasdissolved in PBS and blocking buffer and then added to the peptidecoated plates for 2 hours. Soluble peptides were added to thecorresponding wells to serve as a competition control indicating thespecificity of peptide binding to p53 (+comp) and p53-RE DNA oligo wasadded to other wells (+DNA) to examine whether it affects the binding ofpeptides to p53. After removal of recombinant protein, plates werewashed and incubated with the αp53-HRP conjugated Ab to quantify p53levels. Finally a TMB (substrate of HRP) assay was performed and opticaldensity was determined at 450 nm FIG. 10 shows a representativeexperiment performed with the corresponding peptides and antibodies. Asseen, wells were attached with αp53 monoclonal antibodies to serve asinternal controls of the assay; PAb1801 binds both p53 forms asexpected; PAb1620 is specific to WT p53 and PAb240 is more reactive withthe mutant form. The (blocked) wells were not coated with peptides andpep76 is control peptide sequence. As can be seen, most peptides shownin the figure bind with higher affinity to the recombinant WT p53 ascompared to the mutant p53.

The Effect of pCAP on Mut-p53 Binding to its Responsive Elements in LiveCells.

Next, it was examined whether p53 can also bind to chromatin of itstarget genes. Using chromatin immunoprecipitation (ChIP) assay, it wasexamined whether pCAPs can restore the Mut-p53 DNA binding ability top53 response elements (p53-RE). Breast carcinoma BT-549, endogenouslyexpressing mutant p53^(R249S), were treated for 5 hour with a mix of 3pCAPs; 250, 308 and 325. Cells treated with a mix of control peptidesserved as a negative control. Then cells were fixed and DNA was shearedby sonication. The DNA cross-linked to p53 was immunoprecipitated usingpolyclonal anti p53 antibody. DNA was purified and then p53 responsiveelements of different p53 target genes were quantified using differentprimers in the qPCR reaction. Results were normalized to total DNAinput. As a negative control, extracts were immunoprecipitated withbeads without antibody (Beads). As seen in FIG. 11, the binding ofchromatin to the control beads was at a basal level of 0.005% of theinput DNA. pCAP mix did not increase the binding of p53 to anon-specific genomic DNA control sequence, but p53 binding to responsiveelements in PUMA, p21 and CD95 genes was increased 2.34, 9.78 and 4.54fold, respectively, by pCAPs compared to control peptides.

Screening Peptides for Effect on p53 Transcriptional Activity

As additional screening strategy used to identify reactivating peptideswas performed in vivo and is based on a reporter gene assay. It measuresp53 transcriptional activity by quantifying the activity of a reportergene, placed under the control of a promoter containing 17 repeats of ap53 consensus binding site (RGC). The luciferase assay is performed onliving cells and therefore provides an indication on the effect of testpeptides on Mut-p53 function in the context of intact cells. AnRGC-based promoter cloned upstream of a secreted luciferase reporter(TK-RGC-luc) (New England Biolabs (CAT. NO. NO324S)) was used, since itdoes not require lysis of the cells and allows the use of a 96 wellformat.

FIG. 12 shows a representative luciferase assay experiments that wereperformed to assess the ability of peptides to restore transcriptionalactivity to mutant p53. For the in vivo luciferase based screening,H1299 cells were used. Transient transfection of these p53^(−/−) cellswas performed with vectors expressing WT p53, R175H p53, R249S p53 orempty vector as control (Suad, O., et al., Structural basis of restoringsequence-specific DNA binding and transactivation to mutant p53 bysuppressor mutations. J. Mol. Biol., 2009. 385(1): p. 249-65). Cellswere also co-transfected with TK-RGC-luc (CAT. No. NEB, N0324S). 24hours after transfection, cells were treated with the test peptides. 48hours after transfection, a sample of the culture medium was taken forbioluminescence measurements. As can be seen in FIG. 12, in thenon-treated samples, transfection of WT p53, (positive control), inducedtranscription from TK-RGC-luc by 20-30 fold as compared to TK-RGC-lucalone. When examining the peptide treated samples, it is seen that thepeptides had no significant effect on WT p53 activity; this is anencouraging result, since peptides greatly increasing WT p53 activityare expected to have toxic effects on normal cells. Two of the testedpeptides, namely pCAP-68 and pCAP-75, induce transcription fromTK-RGC-luc in the presence of R175H p53 and the R249S p53.

Screening Peptides for Effect on Viability of Mutant p53 ExpressingCells

An important indication for the reactivating peptides activity is theireffect in-vivo on cancer cells that express Mut-p53. In particular,reactivating peptides that can cause specifically Mut-p53-dependentdeath of cancer cells, with minimal toxic effects on normal cells aredesired. A crystal-violet based viability assay, in which crystal-violetis employed to stain cells that adhere to the plate and therefore theamount of dye is proportional to cell number was used to determine theeffect of the various test peptides on Mut-p53-dependent death. Thecrystal-violet assay is straightforward, fast, reliable, inexpensive anddoes not require a complicated preparation of samples.

Cells were plated in 96-well plates, at calibrated density that allowsthem to grow for 48 hours without reaching confluence. Peptides areadded 6 hours later. Different concentrations of etoposide (cytotoxicdrug) were used as positive control for cell death and as a standardreference curve to assess the effect of tested peptides. 48 hours aftertreatment, cells were washed with PBS to exclude dead cells and debris,and cells that remained attached to the plate were stained withcrystal-violet for 30 minutes. Crystal-violet was removed and cells werewashed with PBS 4 times to eliminate remains of crystal-violet. Then,the stained cells were dissolved in 10% acetic acid and plates weretaken for optical density measurement at 595 nM (specific tocrystal-violet).

FIGS. 13A and 13B illustrate a representative experiment of screeningperformed on 128 synthesized peptides. In this experiment, WI-38fibroblasts were used. These cells express endogenous WT p53 and werefurther infected with a virus expressing either mouse Noxa shRNA, as anonspecific control or the R175H p53 mutant for stable overexpression ofmutant p53. Both of these sub-lines (mNoxa or R175H p53) were seeded at3000 cells per well and treated as described above. The optical densityreads (595 nm) reflect the number of cells in the plate after treatment,normalized to the non-treated samples that are considered as 100%viable. As seen, although WI-38 cells are relatively resistant tokilling, the increasing concentrations of etoposide serve as a goodpositive control for cell death and growth arrest with the highestconcentration reducing cell number by 50% after 48 hours.

Several of the tested peptides indeed caused a significant reduction incell numbers; this reduction was mutant p53 dependent, since it was muchmore prominent in the R175H p53 expressing cells as compared to mNoxa-icontrol cells. These peptides include, for example, pCAP-36, pCAP-46,pCAP-47, pCAP-60, pCAP-97. On the other hand, some peptides were foundto have a toxic effect on both cell sub-lines; one example is pCAP-68.Similar assay was performed on several different Mut-p53-expressinghuman cancer cell lines, the results for the different peptides aresummarized in Table 7.

Example 11 Homology of Lead Peptides to Sequences of Known p53 BindingProteins

After performing the functional screen of peptide motifs predicted byphage display, 20 peptides were identified that exerted functionaleffects on mutant p53 in a variety of assays and cell lines. Next, thesimilarity of these peptides to sequences of human proteins in generaland to proteins known to interact with p53 in particular was examined,since high similarity to proteins interacting with p53 can serve as anindication to the biological significance of a particular motif and canprovide validation of the assumption that the peptides selected underartificial in-vitro conditions can indeed interact with p53. Moreover,the protein structure and surrounding sequence might be helpful indesigning improved peptides that are based both on selection andrational design. To find similarities between peptide sequences andknown human proteins, the BLAST (Basic Local Alignment Search Tool)algorithm was used. The peptide motifs were introduced as querysequences against a sequence database containing human proteinsequences. BLAST finds sub-sequences in the database that are similar tosubsequences in the query. The main idea of BLAST is that there areoften high-scoring segment pairs (HSP) contained in a statisticallysignificant alignment. BLAST searches for high scoring sequencealignments between the query sequence and sequences in the databaseusing a heuristic approach that approximates the Smith-Watermanalgorithm Based on the similarities between the peptide motifs and knownhuman proteins and structural data of these proteins, a list of newpeptide sequences was designed (shown in Table 8 below), in which aminoacids similar to peptide motifs are flanked by other amino acids derivedfrom the protein sequence either flanking the motif or from structuralelements in physical proximity to the homologous motif according to3-dimensional crystallographic data.

Over 70 different proteins with varying degree of similarity to selectedpeptide motifs were identified. Many of these proteins had been shownpreviously to physically interact with p53, while others were reportedto be involved in the p53 signaling pathway, either upstream ordownstream of p53. Several motifs were found to have a very high degreeof homology to known p53 interacting proteins; pCAP-97 (WNHHHSTPHPAH,SEQ ID NO:10) for example has 100% homology to RAD9A (with a p-value of10⁻⁸) which was shown to interact and activate p53; pCAP-60(SFILFIRRGRLG, SEQ ID NO:302) and pCAP-63 (HNHHHSQHTPQH, SEQ ID NO:226)have 90% homology to GAS2 protein sequence (KILFIRLMHNKH, SEQ ID NO:369)in which these motifs are separated by two amino acids (amino acidssimilar to peptide motifs are highlighted in bold letters).

Several alternative and complementary methods to screen lead peptidecandidates for conformational and functional effects on Mut-p53 wereemployed. For increased penetration of peptide across cell membraneseach peptide contains 3-6 Arginine residues either as part of itssequence or added either at its N-terminus or its C-terminus. 40peptides were also conjugated to myristoil fatty acid (myr) for enhancedfusion with cell membranes that would potentially lead to betterdelivery into cells. In-vitro based assays for evaluation were firstperformed, such as ELISA for assessment of p53 conformation andsequence-specific DNA binding of p53. Subsequently, the peptides'activity was examined in live cells by viability assays, p53transcriptional activity on a luciferase reporter gene, and examinationof p53 target genes in-vivo. Combination of these assays (all performedin a 96 well format) allowed the identification and validation of thepeptides' effects on different p53 activities and their ability toconfer such ability to Mut-p53 proteins. As seen from Table 8, 12peptides were found to have a total activity score above 30; all ofthese 12 peptides were shown to be effective in a variety of differentassays including p53 conformation and sequence-specific DNA binding,reduction in viability of Mut-p53 expressing cells and activation of p53target genes. Some of these lead peptides, which have a core motifderived from phage display with added sequences of known proteins (pCAPs201-326) showed a significantly increased effect compared to peptidesderived from phage display alone (pCAPs 1-180), while others werecomparable to pCAPs 1-180.

After careful examination of peptide sequences that have shown the mostsignificant effect in a combination of the assays, it was found that thelead peptides can be classified into several major groups, according totheir consensus motifs. The consensus motifs consist of at least 3consecutive amino-acids, which hypothetically form a sequential orconformational binding site for p53 mutants. These consensus motifs werefound to be HSTPHP, FPGHTIH, IRGRIIR, LPNPPER, SFILFIR, HANLHHT,YPTQGHL, WNHHHSTPHP, TLYLPHWHRH, YRRLLIGMMW, IRILMFLIGCG, SFILFIRRGRLG,LRCLLLLIGRVG, SWQALALYAAGW, IRILMFLIGCGR, glrgrriflifs, HSSHHHPVHSWN,LRCLLLLIGRVGRKKRRQ (SEQ ID NOs:314, 268, 282, 340, 376, 298, 377, 378,253, 20, 379, 302, 275, 380, 273, 381, 280 and 382, respectively).

Effect of Test Peptides on p53 Target Genes

The WT p53 protein works primarily as a transcription factor. Uponactivation by different forms of stress it is accumulated, binds to itsresponsive elements in many target genes and trans-activates theirtranscription. Proteins that are the products of these target genesexecute their functions; transactivation of p21, for example, leads togrowth arrest, whereas transactivation of PUMA would lead to apoptosis.Therefore one of the most important indications to p53 functionalactivation is the induction of its different target genes. The effect ofvarious test peptides on p53 target genes was therefore tested in-vivo.

For the in vivo functional screening, several experimental systems wereused. One system is based on H1299 cells, which are p53 null and arewidely used for p53 research. H1299 cells stably transfected withMut-p53 (ts) A135V were used. This form of p53 is a temperaturesensitive mutant, which has a mutant conformation at 37° C. and a WTconformation at 32° C. FIG. 14 shows a representative experiment. Inessence, the cells were plated in 12-well dishes, the indicated peptideswere added directly to the medium at a concentration of 5 ug/ml, andcells were then either moved to 32° C. or returned to 37° C. 18 hourslater cells were harvested, followed by extraction of RNA, cDNAsynthesis and real time PCR analysis. The expression level of 3representative p53 target genes was examined; p21, PUMA and Mdm2.Expression of genes in H1299-ts at 37° C. is considered as backgroundlevel and all results are normalized to it, and also to the GAPDHhousekeeping gene. Expression of genes in H1299-ts at 32° C. representsWT p53 conformation and therefore serves as a positive control. As canbe seen, temperature shift to 32° C. greatly increased expression of all3 target genes.

As seen in FIG. 14, the negative control peptide pCAP-76 did not causeinduction of p53 targets. Several tested peptides indeed caused asignificant increase in the expression of p21, PUMA and Mdm2. This wasthe case for pCAP-130, pCAP-135, pCAP-142, pCAP-144 and pCAP-148. Thesepeptides induced transcription of target genes by 2-4 fold, compared to9-11 fold of the positive control, authentic wild type p53. The factthat treatment with peptides induced all three genes but had no effecton expression of these genes in control H1299 (p53^(−/−)) cells impliesthat this induction is p53 dependent.

Since delivery of peptides is a major obstacle in their use astherapeutic agents, different approaches were taken overcome thisobstacle. First, based on the tested lead sequences, short peptidesequence motifs (up to 6 amino acids) were elucidated and synthesized,since these small peptides could cross cell membranes by diffusion. Asecond approach was to synthesize tested peptides with a polyarginineC-terminal tail to facilitate their active uptake by endocytosis-basedmechanisms.

Addition of a poly arginine tail to peptides dramatically increases thesolubility of peptides with a high content of hydrophobic amino acids.In some cases it also significantly increased the activity of thepeptides both in-vitro and in-vivo; pCAP-25 for example was insoluble inDMSO at a concentration of 10 mg/ml and showed no effect on p53 activitywhen tested either for conformational change or viability. WhereaspCAP-68 which has the same amino acid sequence with the addition of the9R tail caused a significant shift in Mut-p53 conformation towardsPAb1620, as well as massive cell death. Lead peptides were furthersubjected to rigorous examination of effects on cell viability in aMut-p53 specific manner.

Experiments using different cancer cell lines expressing endogenouslydifferent p53 mutant isoforms were performed. FIGS. 15A and 15Billustrate two representative experiments performed on MDA-MB-231 (FIG.15A) and SKBR3 (FIG. 15B) breast cancer cells expressing Mut-p53 withmutations at positions 280 or 175, respectively, within the DNA BindingDomain (DBD). To examine the peptides' specificity for Mut-p53, thecontrol used was such cells with a knockdown of Mut-p53 (shp53). As seenin FIGS. 15A and 15B, many of the tested peptides showed a reduction incell viability in a Mut-p53 specific manner, with significant readingsof 30%-80% relative to the 100% viability represented by non-treatedMut-p53-expressing cells. Some peptides show some degree of a toxiceffect on cell viability in general, as seen in the shp53 cells. Forexample pCAP-155 exhibited a 30% to 40% reduction in viability in thetwo shp53 infected cell sublines. Furthermore, it is also seen that somepeptides show specific reduction in cell numbers in particular celltypes compared to minimal activity in others. pCAP-146 for examplecaused a significant decrease in MDA-MB-231 shCon cells but almost nospecific effect on SKBR3 shCon cells.

The tested peptides were further tested for their effect on p53 targetgene expression in SKBR3 cells expressing endogenous R175H p53. Theresults are shown in FIG. 16, which shows a bar graph of arepresentative experiment performed on SKBR3 ShCon cells and SKBR3 Shp53cells, knocked down for p53 expression. In essence, these cells wereplated in 12-well dishes; the indicated peptides were added directly tothe medium at a concentration of 5 ug/ml. 18 hours later cells wereharvested, followed by qRT-PCR analysis. Expression level of p21, PUMAand Mdm2 was evaluated. Expression of those genes in non-treated cellsis considered background and all results were normalized to it, as wellas to GAPDH. As seen, some of the lead peptides exhibited a significanttransactivation of p53 target genes. This effect was mediated throughMut-p53 since it was not observed in SKBR3 shp53 cells. pCAP-155,pCAP-144 and pCAP-148 showed among the highest transactivation levels.

Effect of Test Peptides on Apoptosis and Correlation to Activation ofp53 Target Genes

FIGS. 17A and 17B illustrate a representative experiments performed onES2 ovarian carcinoma cells (FIGS. 17A-D) expressing Mut-p53 with amutation at positions 241 within the DBD. Briefly, the cells were platedin 6 cm dishes, and the indicated peptides were added directly to themedium at a concentration of 12 ug/ml at the indicated time points.Cells were harvested, and 60% of the cells were taken for Annexin-PIapoptosis assay and 40% for extraction of RNA, cDNA synthesis and realtime PCR analysis. Apoptosis was assayed using the Annexin-V stainingkit (Roche, REF 11 988 549 001). Non-fixed cells were stained with bothanti Annexin FITC conjugated antibody to detect apoptotic cells, and PI(propidium-iodide) to stain dead cells permeable to the compound,according to the manufacturer's instructions. Stained cells were thenanalyzed by flow cytometry. A total of 10,000 cells was counted for eachsample and divided into four subpopulations according to stainingintensity: cells negative for both PI and Annexin (−PI, −Annexin) aretermed live; cells negative for PI and positive for Annexin (−PI,+Annexin) are going through early stages of apoptosis; cells positivefor PI and Annexin (+PI, +Annexin) are dead cells that underwent anapoptotic process; and cells positive for PI and negative for Annexin(+PI, −Annexin) are assumed to be dead cells that died a non-apoptoticdeath such as necrosis. As seen in the FIGS. 17A, 17B non-treated cells(time 0 h) are mostly (94%) negative for both PI and Annexin, meaningthat the cells are viable and well. Treatment with pCAPs 242 and 250causes a rapid increase in apoptotic cells followed by cell death andafter 5 hours of treatment 12% of cells are Annexin positive and about7% are dead. After 16 and 24 hours of treatment with pCAP 250 theapoptotic population increases to about 27% and dead cells accumulate to29% at 16 h and 36% after 24 h. This trend is true for pCAP 242 as well,although its effects are attenuated and slower. The effect of peptideson cell viability is accompanied by significant transactivation of p53target genes as seen in FIGS. 17C and 17D, which show the expression of4 representative targets. As seen all the genes are activated followingpeptide treatment, and p21 and PUMA mRNA expression increase over timeup to 10 fold and 6 fold following treatment with pCAP 250 and pCAP 242,respectively. CD95 and Btg-2 expression is elevated up to 6 fold overnon-treated cells.

Example 12 In-vivo (preclinical) testing of Mut-p53 reactivatingpeptides

The in-vivo (preclinical) experiments were performed in two types ofmodels: human xenograft models in nude mice and Mut-p53 “knock-in” mice.In each model, the effects of intratumoral injection of the testedpeptides on tumor growth and animal survival are determined.

In the xenograft preclinical model, tumor cells are transfected with aluciferase expression vector, allowing tumor monitoring by live imaging.

In the Mut-p53 “knock-in” mice model a lung specific conditional Mut-p53knock-in mouse is used (Kim, C F, et al., Mouse models of humannon-small-cell lung cancer: raising the bar. Cold Spring Harb. Symp.Quant. Biol., 2005. 70: p. 241-50. Olive, K. P., et al., Mutant p53 gainof function in two mouse models of Li-Fraumeni syndrome. Cell, 2004.119(6): p. 847-60). This model offers a compound conditional knock-inmice with mutations in K-ras combined with one of three p53 alleles:R273H, R175H, or a p53-null allele. Infection with AdenoCre inducesrecombination of the conditional alleles and was shown to produceK-ras-induced lung adenocarcinomas as early as 6 weeks after tumorinitiation. This model closely recapitulates several aspects of advancedhuman pulmonary adenocarcinoma and it allows for two different mutants(175 and 273) to be expressed from the endogenous p53 promoter, atphysiological levels, with the correct spatial and temporal profile.This model allows to demonstrate the features of the tested reactivatingpeptides, in vivo, with respect to several crucial; safety-negligibleeffect on normal mouse tissue or non-infected mice; efficacy-reductionin tumor size and number in treated mice compared to the control; andspecificity to tumor reduction in Mut-p53 expressing mice compared top53 knock out mice. In addition, dose escalation experiments areperformed with positive control peptides, to evaluate the minimal activeconcentrations and the maximal tolerated dose.

Preclinical Trials in a Xenograft Model

MDA-MB-231 cells endogenously expressing p53 R280K were infected with aluciferase expression vector and either shp53 for p53 knockdown or mouseNOXA shRNA (shmNOXA) as a nonspecific control. MDA-MB-231 cells arehighly tumorigenic, forming aggressive, fast growing tumors, as well asbeing metastatic in humans. In total 10 mice were injected. Each mousewas injected subcutaneously with 2*10⁶ MDA-MB-231 cells expressing shp53in the right flank, and with 2*10⁶ MDA-MB-231 cells expressing shmNOXAon the left side. Tumors were allowed to grow for 14 days in order toreach visible size. Growth was monitored by live imaging, using theIVIS200 system. In this system, luciferase bioluminescence isproportional to cancer cell number. The results are presented in FIGS.18A to 18C and FIGS. 19A to 19C. 14 days post injection of the cells, 4mice (mice 7-10) were assigned to the control group (FIGS. 18A to 18C)and 6 mice (mice 1-6) were assigned to the treatment group (FIGS. 19A to19C). Control treatment was composed of a mixture of 3 control peptides(pCAPs 76, 77 and 12), which showed no effect (phenotype) on p53 invitro. The treatment group mice were injected with a mixture of 3peptides (pCAPs 174, 155 and 159) that showed the best phenotypiceffects in vitro on p53. pCAP-159 (SEQ ID NO:312) has a similar sequenceto pCAP-60 (SEQ ID NO:302) with the addition of arginine residues, thepeptide is composed of D-amino acids and is synthesized in the reverseorder (pCAP-159: rrrrrrrrglrgrriflifs (SEQ ID NO:312)) compared topCAP-60: SFILFIRRGRLG, (SEQ ID NO:302) (lowercase letters stand forD-amino acids), in a “retro-inverso” strategy. Peptides were injecteddirectly into the tumor (intra-tumoral injection) three times a week ina volume of 40 μl per tumor and a concentration of 50 μg/ml for eachpeptide in the mix. Therefore a total of 6 μg mix of either the controlpeptides or the treatment peptides was administered each time to eachmouse. The mice were monitored for a total of 5 weeks from the start ofthe peptide treatment. Bioluminescence was measured every 7 days. Asshown in FIG. 18A, shmNOXA tumors, expressing endogenous Mut-p53, showeda 6-15 fold (logarithmic scale) increase in luciferase intensity overthe time-course of the experiment when treated with the control peptidemix. Mouse 10 had to be sacrificed after 28 days of treatment since thetumors reached a limiting large size. FIG. 19A shows the analysis ofmice treated in parallel with a mixture of 3 Mut-p53-activatingpeptides. As seen in FIG. 19A, none of the tumors showed a significantincrease in number of cancer cells over the 35 day period of theexperiment. Two of the tumors (mouse-1 and mouse-4) showed a partialresponse to treatment, evident as a reduction of 50% to 65%,respectively, in bioluminescence. Mice number 2 and 5 showed a completeresponse, with luciferase readings that were as low or close tobackground threshold detection levels of the IVIS system (5*10⁶ photons)even after 21 days of treatment. Administration of peptides wasdiscontinued after 35 days, and mice number 2 and 5 were left withoutany further treatment and monitored for another 21 days. No tumorreappearance was detected in those mice either visually or by liveimaging.

Preclinical Trial #2

MDA-MB-231 cells endogenously expressing p53 R280K were infected with aluciferase expression vector 15 mice were injected subcutaneously with1×10⁶ MDA-MB-231-luc cells on both hips. Tumors were allowed to grow for10 days in order to reach visible size and from that time point onwardstumor growth was monitored by live imaging. The results are presented inFIGS. 20A to 20C. 18 days post injection of the cells, 6 mice wereassigned to the control group and 9 mice were assigned to the treatmentgroup. As before, control treatment involved a mixture of 3 controlpeptides (pCAPs 76, 77 and 12). The treatment group mice were injectedwith a mixture of 3 peptides (pCAPs 174, 155 and 159). Peptides wereinjected directly into the tumor (intra-tumoral injection) three times aweek in a volume of 40 μl per tumor and a concentration of 50 μg/ml foreach peptide in the mix. As shown in FIGS. 20A-D, both the control andthe treatment group showed a similar behavior before treatment; about2-3 fold (logarithmic scale) increase in luciferase intensity (day10-18). FIG. 20A shows the analysis of mice treated in parallel with amixture of 3 control peptides: as seen, the control treatment has only avery mild effect on tumor growth, reducing the rate of growth whencompared to the period before treatment. However, as seen in FIG. 20B,treatment with the mixture of three p53 reactivating pCAPs caused asignificant decrease in the luminescence of the MDA-MB-231 tumors. Aftera single injection of pCAP mix, the average luminescence was reduced by70% and 7 out of the 18 tumors showed total regression with live imagingreadings close to the background detection threshold (data not shown).As shown in FIG. 20B, 12 days after beginning of treatment (4injections) the average tumor luminescence was decreased by 93%, and 11out of 18 tumors showed a complete response. Only one of the 18 tumorsshowed either no or a week response. This tumor was relatively bigbefore beginning of treatment, therefore it is possible that the pCAPdose was not sufficient to cause a significant response.

Preclinical Trial #3—SW-480 Colon Carcinoma Cells

After observing the highly significant result in the MDA-MB-231experiment, additional studies were aimed to extend the observation andexamine cells from a different origin, harboring a different p53 pointmutation. The SW-480 colon carcinoma cell line harbors two endogenousp53 mutations: the R273H and P309S. SW-480 cells were stably infectedwith the luciferase reporter gene, and 10⁶ cells were subcutaneouslyinjected into nude mice. The experiment contained 15 mice that wererandomly divided during the experiment into 3 groups: a control grouptreated with a cocktail of 3 pCAPs previously proven ineffective, agroup treated with a cocktail of 3 effective pCAP (250, 308, 325) andfinally a group treated with a single peptide, the pCAP-325. Theduration of the SW-480 experiment was 42 days from the point of cellimplantation. The time line is relative to the first day of treatmentwhich is marked as day 0. FIGS. 21A-E show tumor growth over time in allthree groups as measured by live imaging in the IVIS. As seen, over timethe control tumors show an average increase of 2.75 fold in tumor size(as inferred from the change in the log of luminescence intensity meanfrom 9.24 at day 0 to 9.68 at day 35, presented in FIG. 21A). The tumorsin the mix treatment group show a decrease equivalent to a 96.7% tumorsize decrease (as inferred from the change in log of luminescenceintensity mean from 9.13 at day 0 to 7.65 at day 35, presented in FIG.21B). Similarly, the tumors in the pCAP 325 group showed an average foldchange of 0.043 which is equivalent to a 95.6% tumor size decrease (asinferred from the changed in log of luminescence intensity mean from8.97 at day 0 to 7.61 at day 35, presented in FIG. 21C).

Summary of Preclinical Experiments

4 pre-clinical experiments have already been performed thus far, usingthe xenograft model of Mut-p53 expressing cells transfected with aluciferase expression vector, allowing tumor monitoring by live imaging.Two experiments were performed with MDA-MB-231 triple negative breastcancer cells (p53 R280K), one experiment used SW-480 colon cancercarcinoma cells (p53 R273H) and another experiment used SKBR3 breastcancer cells (p53 R175H). In each experiment, cells from thecorresponding cell line were injected subcutaneously and allowed theformation of well-established tumors visible both by eye and by liveimaging (typically 2-3 weeks). A treatment regimen was thenadministered, composed of intra-tumor injection of either effective leadpeptides or control peptides (showing no activity in-vitro) every threedays for a period of up to 42 days.

In all pre-clinical experiments performed, mice treated with leadpeptides have shown a very significant decrease in all of their tumorparameters (percentages vary among different experiments); meanluminescence signal (81%-99% as measured by IVIS), tumor weight andvolume (72%-93% measured after tumor extraction). The tumors of micetreated with control peptides on the other hand, continued to grow,although at a reduced rate compared to growth rate before treatment.Almost all of the tumors treated with lead peptides responded totreatment, and 35%-70% of treated tumors showed a complete response withtumors regressing to below threshold detection levels. Six of the miceshowing complete response were kept alive for two months aftercompletion of the experiment (without treatment) and no recurrence oftumors was detected.

In-Vivo Testing of Toxicity of Peptides

In total, 6 mice were used to test toxicity of the peptide mix: two micefor each peptide concentration. The peptide mix used in this experimentwas the same as that described above (FIGS. 19A to 19C) (pCAPs 174, 155and 159). Mice were injected intraperitoneally, three times a week forthree weeks, with a peptide mix prepared at a concentration of 100ug/ml. Two mice were injected with a volume of 40 μl resembling thetotal amount received by mice in the preclinical testing. Two mice wereinjected with 120 μl, and the remaining two mice were injected with 400μl. Given that the average weight of a mouse is 20 g, these amountsrepresent concentrations of 0.6, 1.8 and 6 mg/Kg, respectively. The micewere inspected daily after injection. No visible change was detected inany of the mice. Furthermore, the tissue surrounding the tumors of miceused in the preclinical experiment (FIGS. 18A to 18C and FIGS. 19A to19C) was examined after the mice were sacrificed, for signs of necrosisor inflammation. However, the tissue surrounding the tumor appearednormal in all cases, indicating no major toxic effect of the treatmentwith the pCAP peptides.

Table 10 summarizes the activity of peptides tested in the presentinvention.

TABLE 1 Gene Forward primer Reverse primer p53 CCCAAGCAATGGATGATTTGAGGCATTCTGGGAGCTTCATCT (SEQ ID NO: 343) (SEQ ID NO: 344) p21GGCAGACCAGCATGACAGATT GCGGATTAGGGCTTCCTCTT (SEQ ID NO: 345)(SEQ ID NO: 346) PUMA GACCTCAACGCACAGTACGAG AGGAGTCCCATGATGAGATTGT(SEQ ID NO: 347) (SEQ ID NO: 348) MDM2 AGGCAAATGTGCAATACCAACGGTTACAGCACCATCAGTAGGT A (SEQ ID NO: 349) ACAG (SEQ ID NO: 350) Wig1CGGCAGAGAATTCCACGTGAT ATCTCTTCGCCAGCTCCAACA (SEQ ID NO: 351)(SEQ ID NO: 352) Noxa GCAGAGCTGGAAGTCGAGTGT AAGTTTCTGCCGGAAGTTCAG(SEQ ID NO: 353) (SEQ ID NO: 354) Fas receptor ACTGTGACCCTTGCACCAAATGCCACCCCAAGTTAGATCTGG (SEQ ID NO: 355) (SEQ ID NO: 356) BTG2AGGCACTCACAGAGCACTACA GCCCTTGGACGGCTTTTC (SEQ AAC (SEQ ID NO: 357)ID NO: 358) GAPDH ACCCACTCCTCCACCTTTGA CTGTTGCTGTAGCCAAATTCGT(SEQ ID NO: 359) (SEQ ID NO: 360)

TABLE 2 Selection for R175H p53. Selection round selection marker Titerof phage 1 1620Ab + R175Hp53 100   2 1620Ab + R175Hp53 10⁵ 2 1620Ab5*10⁴ 3 1620Ab + R175Hp53 10⁶ 3 1620Ab 2*10⁶

TABLE 3 Alternating selection for Mut-p53 and WT p53. Selection roundThe selection marker Titer of phage 1 PAb1620 + p53 R175H 2*10² 2 His-WTp53 + Ni 2*10³ 3 PAb1620 + p53 R175H 10⁵ 3 PAb1620 4*10³

TABLE 4 Se- lec- tion # Library round Selection type Titer 1 phd-7 1°1620 + 175 2*10² 31 phd-12 1° 1620 + 175 2*10³ 32 phd-7 1° Tag--wt 1*10³81 phd-12 1° Tag--wt 5*10³ 4 phd-7 1° RE--wt 1*10³ 33 phd-7 2° 1620 +175, 1620-wt 2*10⁵ 39 phd-7 2° 1620 + 175, 1620 + 175 1.5*10⁴  47 phd-72° 1620 + 175, 1620 + 175 2*10⁴ 45 phd-7 2° 1620 + 175, 1620-175 2*10⁴52 phd-12 2° Tag-wt, 1620 + 175 1.5*10⁶  41 phd-7 2° 1620 + 175, 16208*10³ 90 phd-12 2° Tag-wt, 1620 + 175 1.5*10⁵  34 phd-7 2° 1620 + 175,Tag-wt 5*10⁴ 40 phd-7 2° 1620 + 175, Tag + 175 3*10⁴ 48 phd-12 2° 1620 +175, Tag + 175 4*10⁴ 40 phd-7 2° 1620-wt, Tag + 175 4*10⁴ 44 phd-12 2°1620 + 175, Tag 1*10³ 51 phd-7 2° 1620-wt, Tag + 175 2*10⁶ 83 phd-7 2°Tag-wt, Tag + 175 2*10⁶ 55 phd-12 2° 1620 + 175, Tag-wt 2*10⁴ 5 phd-7 2°1620 + 175, Ni-wt 3*10⁴ 82 phd-7 2° Tag-wt, Ni-wt 3*10³ 10 phd-12 2°1620 + 175, Ni-wt 1*10⁵ 38 phd-7 2° 1620 + 175, RE-wt 5*10⁵ 53 phd-12 2°1620 + 175, RE-wt 5*10⁴ 86 phd-7 2° Tag-wt, RE-wt 2*10⁵ 91 phd-12 2°Tag-wt, RE-wtDBD 1*10⁵ 35 phd-7 3° 1620 + 175, Ni-wt, 1620-wt 3*10⁵ 42phd-12 3° 1620 + 175, Ni-wt, 1620 + 175 5*10⁴ 64 phd-7 3° 1620 + 175,RE-wt, 1620 + 175 1*10⁶ 36 phd-7 3° 1620 + 175, Ni-wt, Tag-wt 1*10⁶ 43phd-7 3° 1620 + 175, Ni-wt, Tag + 175 2*10⁶ 56 phd-12 3° 1620 + 175,Ni-wt, Tag + 175 2*10⁵ 65 phd-7 3° 1620 + 175, RE-wt, Tag + 175 2*10⁶ 69phd-7 3° Tag-wt, 1620 + 175, Tag + 175 5*10⁶ 85 phd-12 3° 1620 + 175,RE-wt, Tag + 175 2*10⁵ 92 phd-7 3° 1620 + 175, Ni-wt, Tag-wtDBD 3*10⁵ 93phd-7 3° 1620 + 175, Ni-wt, Tag + 175 4*10⁵ 94 phd-12 3° 1620 + 175,Ni-wt, Tag + 249DBD 4*10⁶ 95 phd-7 3° Tag-wt, 1620 + 175, Tag + 1755*10⁶ 98 phd-7 3° 1620-wt, Tag + 175, Tag + 249DBD 5*10⁶ 37 phd-7 3°1620 + 175, Ni-wt, RE-wt 5*10⁵ 24 phd-7 3° 1620 + 175, Ni-wt, RE 5*10²57 phd-12 3° 1620 + 175, Ni-wt, RE-wt 8*10⁴ 75 phd-7 3° Tag-wt, 1620 +175, RE-wt 5*10⁴ 96 phd-7 3° Tag-wt, 1620 + 175, RE + wtDBD 1.5*10⁶  101phd-7 3° Tag-wt, 1620 + 175, RE 1.5*10⁶  97 phd-12 3° 1620 + 175,Tag-wt, RE + wtDBD 5*10⁴ 118 phd-12 3° RE + 249DBD 5*10⁴

TABLE 5 12 aa Library 7 aa Library #Reads Sequence #Repeats #ReadsSequence #Repeats 553571 KPPDRLWHYTQP 177 194006 HFSHHLK 150 (SEQ ID NO:(SEQ ID 322) NO: 152) 71970 NPNTYVPHWMRQ 66 149576 LPNPPER 111(SEQ ID NO: (SEQ ID 19) NO: 328) 68333 ATLPFVTDRQGW 85 119076 LIISKTLV81 (SEQ ID NO: (SEQ ID 323) NO: 329) 60270 FYSHSTSPAPAK 72 96985 H*VHTHQ54 (SEQ ID NO: (SEQ ID 324) NO: 330) 40419 CYSHSYPTQGHL 43 94834 KLQVPIK51 (SEQ ID NO: (SEQ ID 325) NO: 182) 20256 SLLIGFGIIRSR 49 93473 KPDSPRV60 (SEQ ID NO: (SEQ ID 165) NO: 22) 18938 KPPDRLWHYTQP 88385 SSSLGTH 90(SEQ ID NO: (SEQ ID 322) NO: 331) 13261 SLLIGFGIIRSR 85894 HEVTHHW 66(SEQ ID NO: (SEQ ID 165) NO: 332) 13048 EFHSFYTARQTG 11 79729 SAPQPAT 81(SEQ ID NO: (SEQ ID 326) NO: 333) 10943 NHPWQFPNRWTV 7 76099 TPPLTLI 69(SEQ ID NO: (SEQ ID 287) NO: 334) 10914 SLLIGFGIIRSR 73014 TIHPSIS 42(SEQ ID NO: (SEQ ID 165) NO: 258) 8643 GAMHLPWHMGTL 8 68925 HPWTHH 48(SEQ ID NO: (SEQ ID 285) NO: 335) 8622 IPMNFTSHSLRQ 6 51964 SAASDLR 40(SEQ ID NO: (SEQ ID 248) NO: 336) 7072 KPPDRLWHYTQP 43941 SPLQSLK 33(SEQ ID NO: (SEQ ID 322) NO: 337) 6657 SDGFVPHFKRQH 4 39254 RPTQVLH 27(SEQ ID NO: (SEQ ID 327) NO: 338) 6427 SLLIGFGIIRSR 39167 DSLHSTY 24(SEQ ID NO: (SEQ ID 165) NO: 101) 5311 SEFPRSWDMETN 4 36985 WTLSNYL 30(SEQ ID NO: (SEQ ID 24) NO: 100)

TABLE 6 SEQ ID pCAP NO:  NO:  Sequence 17 8 LTFEHYWAQLTS 18 12GGGGGGGGGGGG 19 19 NPNTYVPHWMRQ 20 25 YRRLLIGMMW 21 26 DEFHSFYTARQTG 2229 KPDSPRV 23 31 PPYSQFLQWYLS 24 40 SEFPRSWDMETN 25 45 HDTHNAHVG 26 50WSEYDIPTPQIPP 27 69 SILTLSRRRRRRRRRR 28 73 SCRCRLRGDRGDR 29 76GGGGGGGGGRRRRRRR 30 77 SEYLCSSLDAAG 31 78 GESFVQHVFRQN 32 79SVHHHHRMHLVA 33 84 GRRRFCM 34 85 KLTIHHH 35 86 FGSHHEL 36 96 GTVDHHA 37107 DRLSVFLFIM 38 114 AISHHTR 39 116 KHHPFDHRLGNQ 40 119 HSAHHTM 41 125ELGLHRH 42 126 RRLRICV 43 156 VPHIHEFTRRRRRRRR 44 164 PLTLI 45 165 SLLIG46 166 KPPER 47 168 CRIIR 48 169 SFILI 49 171 PHHHS 50 172 EFHS 51 173RLRRL 52 175 DSPR 53 176 HPWTH 54 177 HFSHH 55 178 RRVI 56 179 ILVI 57207 RRSRSNEDVEDKTEDE 58 208 RRIRSGGKDHAWTPLHE NH 59 209HTPHPPVARTSPLQTPRR 60 211 PDSEPPRMELRRR 61 215 RRDTFDIRILMAF 62 218RREVTELHHTHEDRR 63 223 SPWTHERRCRQR 64 232 RSRSSHLRDHERTHT 65 236RRRSTNTFLGEDFDQ 66 241 LIGLSTSPRPRIIR 67 248 EIYGESGKTDEHALDTEY RR 68252 RRVILRSYDGGHSTPHPD 69 253 TGKTFVKRHLTEFEKKY R 70 254NHFDYDTIELDTAGEYSR RR 71 255 DPEPPRYLPPPPERR 72 260 RRTFIRHRIDSTEVIYQDED 73 262 ESKTGHKSEEQRLRRYR 74 263 YDDEHNHHPHHSTHRRR 75 264RRRREVHTIHQHGIVHSD 76 269 DEPLPPPERRR 77 270 SPHPPY 78 271SPHPPYSPHPPYSPHPPYP 79 272 RRPHNLHHD 80 274 LRDPHPPERRIR 81 283RRPADQISYLHPPER 82 291 DLQYDFPRIRR 83 292 YDELYQKEDPHRRR 84 294FKPERFPQNDRRR 85 296 RPADRIRR 86 297 HDFDPRYRDRR 87 300 RIRRDPDSPLPHPE88 304 myr-RRIRILMFLIGCGRV 89 309 HPHVILPRIRIRIR 90 311 EIHTIHLLPERR 91320 EPSHPRSRYPRTF 92 321 RNIIIRDFIHFSHIDR 93 322 RRIRDPQIK- myrLEIHFSHID94 323 myr-DLHTIHIPRDRR 95 324 SHDFPHREPRPERR 96 219 SYRHYSDHWEDRRR 97 2VWVHDSCHANLQNYRN YLLP 98 4 EHDFEVRGDVVNGRNHQ GPK 99 5 LEVIYMI 100 38WTLSNYL 101 39 DSLHSTY 102 41 WHHRQQIPRPLE 103 64 APSIFTPHAWRQ 104 66THFSHHLKGGGRRQRRR P 105 67 LHSKTLVLGGGRRRRGD R 106 71 WTLSNYLGGRKKRRQRRRR 107 81 VRCIFRGIWVRL 108 98 HSSGHNFVLVRQ 109 110 LFILVFR 110 112TTSHHPK 111 124 VMVLFRILRGSM 112 162 SILT 113 214 RRRESEQRSISLHHHST 114216 myr- HFNHYTFESTCRRRRC 115 217 HSTPHPPQPPERRR 116 224RRKSEPHSLSGGYQTGA D 117 234 HRTGHYTRCRQRCRSRS HNRH 118 243RRCRSILPLLLLSR 119 256 RTLHGRRVILHEGGHSIS DLK 120 266 HHRLSYFIVRRHSTHASR121 293 RRIRIDPQHD 122 299 ILQPDFLIRPE 123 307 HDPRIIRIR 124 52 SPYPIRT125 53 ILVIIQRIM 126 101 IRFILIR 127 102 SSVHHRG 128 103 LRRQLQL 129 113HTTAHTH 130 115 HPHNHTVHNVVY 131 117 DHSKFVPLFVRQ 132 120 SIRTLGRFLIIRV133 123 GLCRIIL 134 127 SPPIRHH 135 201 HPTHPIRLRDNLTR 136 212myr-REEETILIIRRR 137 225 HTIHSISDFPEPPDRRRR 138 228 DEDAAHSTGHPHNSQHRRRR 139 240 TEQHHYIPHRRR 140 251 RLRRVILRSYHE 141 265 EEPDRQPSGKRGGRKRRSR 142 273 RDFHTIHPSISRR 143 276 RRVDIHDGQRR 144 277 DQPYPHRRIR 145 281myr-RDFILFIRRLGRR 146 295 LDLYHPRERR 147 298 RRIRDPLGNEHE 148 303IVEFRIRR 149 312 RRPRIPDYIL 150 314 RSTPHIHEFIRR 151 319 SHDFYPHWMRERIR152 13 HFSHHLK 153 32 TSPLQSLK 154 51 AILTLILRRVIWP 155 94 LRFIDYP 156109 GPIKHHLQHH 157 163 LTLS 158 222 RYEENNGVNPPVQVFES RTR 159 239REGFYGPWHEQRRR 160 285 RRDIIRHNAHS 161 286 HDFHDYLERR 162 305 IREFDPRRIR163 310 RLRCLLLLIGRVGRR 164 6 LGIDEDEETETAPE 165 22 SLLIGFGIIRSR 166 27VHEVTHHWL 167 56 ATPFHQT 168 58 SILPLFLIRRSG 169 72 SCRCRLRRRRRRRRRR 170105 SRIVLGW 171 111 SNIHHQV 172 121 LTLMRLRIIG 173 122 HSYSPYYTFRQH 174167 FILIR 175 205 RCRNRKKEKTECLQKESEK 176 213 RRIKMIRTSESFIQHIVS 177 244RRVSELQRNKHGRKHEL 178 246 RRRLDDEDVQTPTPSEYQN 179 261 RRRQPLPSAPENEE 1807 SPLQTPAAPGAAAGPALSPV 181 18 SHQVHTHHNN 182 37 KLQVPIK 183 74IRGRIIRRKKRRQRRRRGDR 184 82 QIPHRSSTALQL 185 88 SYQTMQP 186 140TDSHSHHRRRRRRRRRRR 187 143 IPMNFTSHSLRQRRRRRRRRR 188 153YWSAPQPATRRRRRRRRRRR 189 220 STTHPHPGTSAPEPATRRR 190 226 DDSDNRIIRYRR191 238 TSPHPSLPRHIYPRR 192 247 RRITEIRGRTGKTTLTYIED 193 249myr-DERTGKTRRYIDTRDIRR 194 275 myr-MTYSDMPRRIITDEDRRR 195 278RRYDTVIDDIEYRR 196 279 RDTIERPEIRR 197 280 myr-RYRRLILEIWRR 198 284myr-RHDTHNAHIRR 199 288 THDFDRLLRIRRR 200 289 RHNHIRPDNQ 201 290RYKEPRITPRE 202 302 LRIEPIRIR 203 306 myr-RLIRIRILM 204 318RPEFHSFHPIYERR 205 91 STTHIHA 206 92 FPHLVSSLTT 207 99 GLHLFTTDRQGW 208132 NHPWQFPNRWTRRRRRR 209 145 HSSHHHPVHSWNRRRRRRR 210 316myr-DIHTIHLPDTHRR 211 10 VAEFAQSIQSRIVEWKERLD 212 49 TRILCIVMM 213 55FLLPEPDENTRW 214 57 LMSNAQY 215 89 SILTLSCRCRLRLWR 216 95 HQIHRNHTY 217106 LIRRCSLQR 218 137 GAMHLPWHMGTRRRRRR 219 202 DEDAKFRIRILMRR 220 245NHITNGGEEDSDCSSRRRRL 221 257 myr-HSSHHHPTVQHRR 222 287 RDFERTIVDI 223313 myr-RRREILHPEFRILYE 224 14 HHFSHHWKT 225 59 FLIRRSG 226 63HNHHHSQHTPQH 227 80 HLHKHHYKDSRM 228 231 HRTQSTLILFIRRGRET 229 315LHFSHIDRR 230 62 YELPHHAYPA 231 133 SLLIGFGIIRSRRRRRRRR 232 135HTDSHPHHHHPHRRRRR 233 147 ATQHHYIKRRRRRRRRRRR 234 129FRSFAIPLVVPFRRRRRRR 235 138 YPTQGHLRRRRRRRRRRRR 236 146HANLHHTRRRRRRRRRRR 237 152 YRRLLIGMRRRRRRRRRRRR 238 233 SHYHTPQNPPSTRRR239 235 RSYSKLLCLLERLRISP 240 3 FWTQSIKERKMLNEHDFEVR 241 15 THFSHHLKH242 90 SCRCRLR 243 139 MHPPDWYHHTPKRRRRRR 244 237 HTIHVHYPGNRQPNPPLILQR245 268 TPSYGHTPSHHRRR 246 301 myr-IRGRIRIIRRIR 247 20 HHPWTHHQRWS 24848 IPMNFTSHSLRQ 249 118 SNHHHRHHTNTH 250 130 EVTFRHSVVRRRRRRRRRRR 251149 FPGHTIHRRRRRRRRRRR 252 34 SILTLSRIVLGWW 253 47 TLYLPHWHRH 254 136SILTLRLRRLRRRRRRRR 255 142 TLYLPHWHRHRRRRRRRRRR 256 43 TDSHSHH 257 11EWKERLDKEFSLSVYQKMKF 258 30 TIHPSIS 259 33 SILTLRLRRLRR 260 44 VPHIHEFT261 9 TIIHREDEDEIEW 262 61 KDLPFYSHLSRQ 263 65 THFSHHLKHRRRRRRRRRR 26493 ATQHHYIK 265 108 IIRGNFLIGGRL 266 131 LPNPPERHHRRRRRRRRRRR 267 158SFILFIRRGRLGRGDR 268 100 FPGHTIH 269 128 CILRLWW 270 206RRRSHSQENVDQDTDE 271 204 MSTESNMPRLIQNDDRRR 272 104 LLRLGLI 273 23IRILMFLIGCGR 274 17 LHSKTLVL 275 24 LRCLLLLIGRVG 276 258 FLIGPDRLIRSR277 16 LPNPPERHH 278 28 HTDSHPHHHHPH 279 160 Fitc- SFILFIRRGRLGRRRRRRRRR280 83 HSSHHHPVHSWN 281 259 myr-RTLIGIIRSHHLTLIRR 282 54 IRGRIIR 283 150IIRGNFLIGGRLRRRRRRRRR 284 170 IRILM 285 35 GAMHLPWHMGTL 286 267KRGGRKRRGGGHRLSYFIRR 287 21 NHPWQFPNRWTV 288 42 MHPPDWYHHTPKH 289 141SWQALALYAAGWRRRRRR 290 161 HNAH 291 210 DEFERYRRFSTSRRR 292 1 EVTFRHSVV293 75 TRILCIVRKKRRQRRRRGDR 294 70 SILTLSRGRKKRRQRRRR 295 151CILRLWWRRRRRRRRRRR 296 46 ASWQALALYAAGW 297 229 myr-PRVLPSPHTIHPSQYP 29887 HANLHHT 299 157 SFILFIRRGRLGRKKRRQRRRP 300 36 YPTQGHLR 301 68YRRLLIGMMWRRRRRRRRRR R 302 60 SFILFIRRGRLG 303 134 IRILMFLIGCGRRRRRRRR304 308 myr-RRICRFIRICRVR 305 155 IRGRIIRRRRRRRRRR 306 203RRRHDSCHNQLQNYDHSTE 307 148 WNHHHSTPHPRRRRRRRRRR 308 282myr-RRPVAPDLRHTIHIPPER 309 317 RRDIHTIHPFYQ 310 97 WNHHHSTPHPAH 311 144SFILFIRRGRLGRRRRRRRRR 312 159 rrrrrrrrglrgrriflifs 313 326myr-RRHNAHHSTPHPDDR 314 174 HSTPHP 315 154 LRCLLLLIGRVGRKKRRQRR 316 221myr-RRKHNKHRPEPDSDER 317 325 myr-RRIRDPRILLLHFD 318 230RKRGKSYAFFVPPSESKERW 319 227 myr-RRKILFIRLMHNKH 320 242myr-RRLIVRILKLPNPPER 321 250 myr-RRHSTPHPD

TABLE 7 Name Peptide Seq Conform DNA Viability Luc PCR AS* pCAP1EVTFRHSVV 2 2 3 3 4 3 3 2 3 5 3 32 pCAP2 VWVHDSCHANLQNYRNYLLP 2 2 pCAP3FWTQSIKERKMLNEHDFEVR 2 3 3 2 2 12 pCAP4 EHDFEVRGDVVNGRNHQGPK 2 2 pCAP5LEVIYMI 2 2 pCAP6 LGIDEDEEIETAPE 2 3 5 pCAP7 SPLQTPAAPGAAAGPALSPV 3 3 6pCAP8 LTFEHYWAQLTS 0 pCAP9 TIIHREDEDEIEW 2 3 3 2 2 3 2 17 pCAP10VAEFAQSIQSRIVEWKERLD 3 3 2 8 pCAP11 EWKERLDKEFSLSVYQKMKF 3 3 3 5 2 16pCAP12 GGGGGGGGGGGG 0 pCAP13 HFSHHLK 1 3 4 pCAP14 HHFSHHWKT 2 2 2 3 9pCAP15 THFSHHLKH 2 2 2 3 3 12 pCAP16 LPNPPERHH 2 3 3 3 3 4 4 2 24 pCAP17LHSKTLVL 2 2 3 3 3 3 4 3 23 pCAP18 SHQVHTHHNN 3 3 6 pCAP19 NPNTYVPHWMRQ0 pCAP20 HHPWTHHQRWS 2 3 3 2 3 13 pCAP21 NHPWQFPNRWTV 4 2 2 2 2 3 3 4 34 29 pCAP22 SLLIGFGIIRSR 3 2 5 pCAP23 IRILMFLIGCGR 1 1 3 3 3 3 4 3 1 22pCAP24 LRCLLLLIGRVG 3 2 2 3 3 3 3 4 23 pCAP25 YRRLLIGMMW 0 pCAP26DEFHSFYTARQTG 0 pCAP27 VHEVTHHWL 2 3 5 pCAP28 HTDSHPHHHHPH 2 2 3 3 3 4 13 3 24 pCAP29 KPDSPRV 0 pCAP30 TIHPSIS 3 2 3 2 2 2 2 16 pCAP31PPYSQFLQWYLS 0 pCAP32 TSPLQSLK 1 3 4 pCAP33 SILTLRLRRLRR 2 2 4 4 4 16pCAP34 SILTLSRIVLGWW 2 4 4 4 14 pCAP35 GAMHLPWHMGTL 2 2 2 4 3 2 3 3 3 43 28 pCAP36 YPTQGHLR 6 3 2 2 3 3 3 5 3 3 3 3 39 pCAP37 KLQVPIK 2 2 2 6pCAP38 WTLSNYL 2 2 pCAP39 DSLHSTY 2 2 pCAP40 SEFPRSWDMETN 0 pCAP41WHHRQQIPRPLE 2 2 pCAP42 MHPPDWYHHTPKH 3 2 4 2 2 3 3 3 3 4 29 pCAP43TDSHSHH 2 3 3 3 4 15 pCAP44 VPHIHEFT 2 3 3 3 2 3 16 pCAP45 HDTHNAHVG 0pCAP46 ASWQALALYAAGW 2 2 2 2 3 3 4 6 2 3 3 2 34 pCAP47 TLYLPHWHRH 2 3 33 3 14 pCAP48 IPMNFTSHSLRQ 2 2 3 3 3 13 pCAP49 TRILCIVMM 5 3 8 pCAP50WSEYDIPTPQIPP 0 pCAP51 AILTLILRRVIWP 2 2 4 pCAP52 SPYPIRT 3 3 pCAP53ILVIIQRIM 3 3 pCAP54 IRGRIIR 3 2 2 4 4 4 4 3 26 pCAP55 FLLPEPDENTRW 4 48 pCAP56 ATPFHQT 3 2 5 pCAP57 LMSNAQY 2 2 4 8 pCAP58 SILPLFLIRRSG 2 3 5pCAP59 FLIRRSG 2 2 3 2 9 pCAP60 SFILFIRRGRLG 2 3 3 3 4 2 3 5 4 3 4 4 40pCAP61 KDLPFYSHLSRQ 2 3 2 2 5 3 17 pCAP62 YELPHHAYPA 5 2 3 10 pCAP63HNHHHSQHTPQH 2 3 2 2 9 pCAP64 APSIFTPHAWRQ 2 2 pCAP65THFSHHLKHRRRRRRRRRR 2 2 2 2 2 4 3 17 pCAP66 THFSHHLKGGGRRQRRRP 2 2pCAP67 LHSKTLVLGGGRRRRGDR 2 2 pCAP68 YRRLLIGMMWRRRRRRRRRRR 4 5 4 3 5 5 42 2 2 3 39 pCAP69 SILTLSRRRRRRRRRR 0 pCAP70 SILTLSRGRKKRRQRRRR 3 3 2 2 33 3 3 5 4 2 33 pCAP71 WTLSNYLGGRKKRRQRRRR 2 2 pCAP72 SCRCRLRRRRRRRRRR 23 5 pCAP73 SCRCRLRGDRGDR 0 pCAP74 IRGRIIRRKKRRQRRRRGDR 3 3 6 pCAP75TRILCIVRKKRRQRRRRGDR 3 3 2 2 5 5 3 4 2 3 32 pCAP76 GGGGGGGGGRRRRRRR 0pCAP77 SEYLCSSLDAAG 0 pCAP78 GESFVQHVFRQN 0 pCAP79 SVHHHHRMHLVA 0 pCAP80HLHKHHYKDSRM 3 3 3 9 pCAP81 VRCIFRGIWVRL 2 2 pCAP82 QIPHRSSTALQL 3 3 6pCAP83 HSSHHHPVHSWN 4 1 5 4 4 3 3 1 23 pCAP84 GRRRFCM 0 pCAP85 KLTIHHH 0pCAP86 FGSHHEL 0 pCAP87 HANLHHT 3 2 2 3 3 4 4 3 5 4 4 37 pCAP88 SYQTMQP2 4 6 pCAP89 SILTLSCRCRLRLWR 2 4 2 8 pCAP90 SCRCRLR 2 4 4 2 12 pCAP91STTHIHA 4 3 7 pCAP92 FPHLVSSLTT 4 3 7 pCAP93 ATQHHYIK 4 4 2 3 4 17pCAP94 LRFIDYP 4 4 pCAP95 HQIHRNHTY 4 4 8 pCAP96 GTVDHHA 0 pCAP97WNHHHSTPHPAH 3 3 6 4 4 5 4 4 4 2 3 5 47 pCAP98 HSSGHNFVLVRQ 2 2 pCAP99GLHLFTTDRQGW 2 3 2 7 pCAP100 FPGHTIH 5 3 3 2 3 3 19 pCAP101 IRFILIR 3 3pCAP102 SSVHHRG 3 3 pCAP103 LRRQLQL 3 3 pCAP104 LLRLGLI 3 3 6 3 3 3 21pCAP105 SRIVLGW 3 2 5 pCAP106 LIRRCSLQR 3 5 8 pCAP107 DRLSVFLFIM 0pCAP108 IIRGNFLIGGRL 3 3 2 3 3 3 17 pCAP109 GPIKHHLQHH 2 2 4 pCAP110LFILVFR 2 2 pCAP111 SNIHHQV 3 2 5 pCAP112 TTSHHPK 2 2 pCAP113 HTTAHTH 33 pCAP114 AISHHTR 0 pCAP115 HPHNHTVHNVVY 3 3 pCAP116 KHHPFDHRLGNQ 0pCAP117 DHSKFVPLFVRQ 3 3 pCAP118 SNHHHRHHTNTH 2 2 3 3 3 13 pCAP119HSAHHTM 0 pCAP120 SIRTLGRFLIIRV 3 3 pCAP121 LTLMRLRIIG 2 3 5 pCAP122HSYSPYYTFRQH 2 3 5 pCAP123 GLCRIIL 3 3 pCAP124 VMVLFRILRGSM 2 2 pCAP125ELGLHRH 0 pCAP126 RRLRICV 0 pCAP127 SPPIRHH 3 3 pCAP128 CILRLWW 3 2 3 33 3 2 19 pCAP129 FRSFAIPLVVPFRRRRRRR 4 3 2 2 11 pCAP130EVTFRHSVVRRRRRRRRRRR 6 3 4 13 pCAP131 LPNPPERHHRRRRRRRRRRR 3 3 3 3 2 3 118 pCAP132 NHPWQFPNRWTRRRRRR 3 4 7 pCAP133 SLLIGFGIIRSRRRRRRRR 2 2 6 10pCAP134 IRILMFLIGCGRRRRRRRR 4 4 3 2 6 6 3 3 3 3 3 40 pCAP135HTDSHPHHHHPHRRRRR 3 3 2 2 10 pCAP136 SILTLRLRRLRRRRRRRR 3 3 5 3 14pCAP137 GAMHLPWHMGTRRRRRR 3 3 2 8 pCAP138 YPTQGHLRRRRRRRRRRRR 3 4 2 2 11pCAP139 MHPPDWYHHTPKRRRRRR 2 3 2 2 3 12 pCAP140 TDSHSHHRRRRRRRRRRR 3 3 6pCAP141 SWQALALYAAGWRRRRRR 6 3 5 3 3 3 3 3 29 pCAP142TLYLPHWHRHRRRRRRRRRR 6 2 3 3 14 pCAP143 IPMNFTSHSLRQRRRRRRRRR 2 4 6pCAP144 SFILFIRRGRLGRRRRRRRRR 2 2 2 2 3 3 3 3 3 2 6 6 4 4 4 49 pCAP145HSSHHHPVHSWNRRRRRRR 3 2 2 7 pCAP146 HANLHHTRRRRRRRRRRR 3 2 3 3 11pCAP147 ATQHHYIKRRRRRRRRRRR 4 3 3 10 pCAP148 WNHHHSTPHPRRRRRRRRRR 4 4 45 3 4 2 3 3 3 4 4 43 pCAP149 FPGHTIHRRRRRRRRRRR 3 5 3 2 13 pCAP150IIRGNFLIGGRLRRRRRRRRR 4 4 4 4 3 2 5 26 pCAP151 CILRLWWRRRRRRRRRRR 5 4 54 3 3 3 3 3 33 pCAP152 YRRLLIGMRRRRRRRRRRRR 5 3 3 11 pCAP153YWSAPQPATRRRRRRRRRRR 3 3 6 pCAP154 LRCLLLLIGRVGRKKRRQRR 6 4 5 4 5 5 5

5 3 4 5 3 57 pCAP155 IRGRIIRRRRRRRRRR 3 2 2 5 5 5 4 4 4 3 41 pCAP156VPHIHEFTRRRRRRRR 0 pCAP157 SFILFIRRGRLGRKKRRQRRRP 1 1 1 2 3 3 3 3 5 2 25 2 4 37 pCAP158 SFILFIRRGRLGRGDR 1 1 4 4 4 2 2 18 pCAP159rrrrrrrrglrgrriflifs 3 1 1 1 3 5 4 4 5 4 5 4 5 4 4 53 pCAP160Fitc-SFILFIRRGRLGRRRRRRRRR 1 1 4 4 4 4 3 3 24 pCAP161 HNAH 3 2 2 2 3 5 35 4 29 pCAP162 SILT 2 2 pCAP163 LTLS 4 4 pCAP164 PLTLI 0 pCAP165 SLLIG 0pCAP166 KPPER 0 pCAP167 FILIR 5 5 pCAP168 CRIIR 0 pCAP169 SFILI 0pCAP170 IRILM 5 5 5 3 3 3 3 27 pCAP171 PHHHS 0 pCAP172 EFHS 0 pCAP173RLRRL 0 pCAP174 HSTPHP 1 4 4 5 4 5 4 5 4 4 5 1 4 4 54 pCAP175 DSPR 0pCAP176 HPWTH 0 pCAP177 HFSHH 0 pCAP178 RRVI 0 pCAP179 ILVI 0 *AS -Activity Score.

TABLE 8 Name Sequence Similarity Conformation DNA binding ViabilityPCR 1 AS* pCAP201 HPTHPIRLRDNLTR 14-3-3 3 3 pCAP202 DEDAKFRIRILMRR APAF13 2 3 8 pCAP203 RRRHDSCHNQLQNYDHSTE ASPP1 3 3 2 4 6 4 6 6 4 4 42 pCAP204MSTESNMPRLIQNDDRRR ASPP2 2 4 4 5 5 20 pCAP205 RCRNRKKEKTECLQKESEK ATF3 23 5 pCAP206 RRRSHSQENVDQDTDE BAK 2 3 3 2 1 2 6 19 pCAP207RRSRSNEDVEDKTEDE BAK 0 pCAP208 RRIRSGGKDHAWTPLHENH BARD1 0 pCAP209HTPHPPVARTSPLQTPRR BCL2 0 pCAP210 DEFERYRRFSTSRRR BCL-XL 3 2 2 2 2 6 3 36 29 pCAP211 PDSEPPRMELRRR BCR 0 pCAP212 myr-REEETILIIRRR BRG1 3 3pCAP213 RRIKMIRTSESFIQHIVS BTF 2 3 5 pCAP214 RRRESEQRSISLHHHST C-ABL 2 2pCAP215 RRDTFDIRILMAF CARM1 0 pCAP216 myr-HFNHYTFESTCRRRRC CAS 2 2pCAP217 HSTPHPPQPPERRR CCDC8 2 2 pCAP218 RREVTELHHTHEDRR CEP72 0 pCAP219SYRHYSDHWEDRRR CETD2 1 1 pCAP220 STTHPHPGTSAPEPATRRR CHD6 2 2 2 6pCAP221 myr-RRKHNKHRPEPDSDER CTF2 3 5 3 4 4 2 5 6 5 2 5 4 6 6 60 pCAP222RYEENNGVNPPVQVFESRTR CUL7 4 4 pCAP223 SPWTHERRCRQR CYP27B1 0 pCAP224RRKSEPHSLSGGYQTGAD DIABLO 2 2 pCAP225 HTIHSISDFPEPPDRRRR DMP1 3 3pCAP226 DDSDNRIIRYRR G3BP2 3 3 6 pCAP227 myr-RRKILFIRLMHNKH GAS2 4 6 5 55 5 6 5 6 3 5 5 3 4 67 pCAP228 DEDAAHSTGHPHNSQHRRRR HIPK1 3 3 pCAP229myr-PRVLPSPHTIHPSQYP HIPK2 2 4 4 4 3 3 4 4 4 4 36 pCAP230RKRGKSYAFFVPPSESKERW HMGB1 3 5 5 3 4 3 6 5 5 2 6 4 6 6 63 pCAP231HRTQSTLILFIRRGRET HTRA2 3 6 9 pCAP232 RSRSSHLRDHERTHT HZF 0 pCAP233SHYHTPQNPPSTRRR IFI16 3 3 3 2 11 pCAP234 HRTGHYTRCRQRCRSRSHNRH KLF4 2 2pCAP235 RSYSKLLCLLERLRISP MIF 2 5 2 2 11 pCAP236 RRRSTNTFLGEDFDQMORTALIN 0 pCAP237 HTIHVHYPGNRQPNPPLILQR MULE 3 2 5 2 12 pCAP238TSPHPSLPRHIYPRR NFAT 2 4 6 pCAP239 REGFYGPWHEQRRR OGA 2 2 4 pCAP240TEQHHYIPHRRR OSGIN2 3 3 pCAP241 LIGLSTSPRPRIIR PAR3 0 pCAP242myr-RRLIVRILKLPNPPER PARC 4 3 6 6 6 6 6 3 4 6 6 5 6 67 pCAP243RRCRSILPLLLLSR PERP 2 2 pCAP244 RRVSELQRNKHGRKHEL PIAS1 3 2 5 pCAP245NHITNGGEEDSDCSSRRRRL PIN1 3 3 2 8 pCAP246 RRRLDDEDVQTPTPSEYQN PIRH2 3 25 pCAP247 RRITEIRGRTGKTTLTYIED RAD51 3 3 6 pCAP248 EIYGESGKTDEHALDTEYRRRAD51 0 pCAP249 myr-DERTGKTRRYIDTRDIRR RAD51 3 3 6 pCAP250 myr-RRHSTPHPDRAD9 4 6 5 4 5 5 5 5 6 3 6 6 6 6 72 pCAP251 RLRRVILRSYHE RAD9 3 3pCAP252 RRVILRSYDGGHSTPHPD RAD9 0 pCAP253 TGKTFVKRHLTEFEKKYR RAN 0pCAP254 NHFDYDTIELDTAGEYSRRR RAS 0 pCAP255 DPEPPRYLPPPPERR RASSF5 0pCAP256 RTLHGRRVILHEGGHSISDLK RPA70 2 2 pCAP257 myr-HSSHHHPTVQHRR SIN3A4 4 8 pCAP258 FLIGPDRLIRSR SIVA 6 4 5 2 2 4 23 pCAP259myr-RTLIGIIRSHHLTLIRR SMG1 4 5 4 5 3 2 2 25 pCAP260 RRTFIRHRIDSIEVIYQDEDSTK11 0 pCAP261 RRRQPLPSAPENEE STK15 2 3 5 pCAP262 ESKTGHKSEEQRLRRYR TBP0 pCAP263 YDDEHNHHPHHSTHRRR TSC22 0 pCAP264 RRRREVHTIHQHGIVHSD TTK 0pCAP265 EEPDRQPSGKRGGRKRRSR TWIST 3 3 pCAP266 HHRLSYFIVRRHSTHASR TWIST 22 pCAP267 KRGGRKRRGGGHRLSYFIRR TWIST 3 2 4 6 2 3 2 2 2 2 28 pCAP268TPSYGHTPSHHRRR WT1 3 5 4 12 pCAP269 DEPLPPPERRR 0 pCAP270 SPHPPY 0pCAP271 SPHPPYSPHPPYSPHPPYP 0 pCAP272 RRPHNLHHD 0 pCAP273 RDFHTIHPSISRR3 3 pCAP274 LRDPHPPERRIR 0 pCAP275 myr-MTYSDMPRRIITDEDRRR ASPP2 3 3 6pCAP276 RRVDIHDGQRR 3 3 pCAP277 DQPYPHRRIR 3 3 pCAP278 RRYDTVIDDIEYRR 33 6 pCAP279 RDTIERPEIRR 3 3 6 pCAP280 myr-RYRRLILEIWRR 3 3 6 pCAP281myr-RDFILFIRRLGRR 3 3 pCAP282 myr-RRPVAPDLRHTIHIPPER LTA 4 3 3 4 4 6 2 24 4 4 3 43 pCAP283 RRPADQISYLHPPER 0 pCAP284 myr-RHDTHNAHIRR 6 6 pCAP285RRDIIRHNAHS 4 4 pCAP286 HDFHDYLERR 4 4 pCAP287 RDFERTIVDI 4 4 8 pCAP288THDFDRLLRIRRR 2 4 6 pCAP289 RHNHIRPDNQ 2 4 6 pCAP290 RYKEPRITPRE 4 2 6pCAP291 DLQYDFPRIRR 0 pCAP292 YDELYQKEDPHRRR 0 pCAP293 RRIRIDPQHD 2 2pCAP294 FKPERFPQNDRRR 0 pCAP295 LDLYHPRERR 3 3 pCAP296 RPADRIRR 0pCAP297 HDFDPRYRDRR 0 pCAP298 RRIRDPLGNEHE 3 3 pCAP299 ILQPDFLIRPE 2 2pCAP300 RIRRDPDSPLPHPE 0 pCAP301 myr-IRGRIRIIRRIR 3 3 6 12 pCAP302LRIEPIRIR 3 3 6 pCAP303 IVEFRIRR 3 3 pCAP304 myr-RRIRILMFLIGCGRV 0pCAP305 IREFDPRRIR 4 4 pCAP306 myr-RLIRIRILM 6 6 pCAP307 HDPRIIRIR 2 2pCAP308 myr-RRICRFIRICRVR CDC25B 6 4 4 6 4 4 2 4 6 40 pCAP309HPHVILPRIRIRIR 0 pCAP310 RLRCLLLLIGRVGRR 4 4 pCAP311 EIHTIHLLPERR 0pCAP312 RRPRIPDYIL 3 3 pCAP313 myr-RRREILHPEFRILYE 2 6 8 pCAP314RSTPHIHEFIRR 3 3 pCAP315 LHFSHIDRR 3 6 9 pCAP316 myr-DIHTIHLPDTHRR 3 4 7pCAP317 RRDIHTIHPFYQ HSD17 5 4 3 4 2 5 1 6 5 5 3 43 pCAP318RPEFHSFHPIYERR 3 3 6 pCAP319 SHDFYPHWMRERIR 3 3 pCAP320 EPSHPRSRYPRTF 0pCAP321 RNIIIRDFIHFSHIDR 0 pCAP322 RRIRDPQIK-myrLEIHFSHID 0 pCAP323myr-DLHTIHIPRDRR 0 pCAP324 SHDFPHREPRPERR 0 pCAP325 myr-RRIRDPRILLLHFDCCT3 4 6 3 4 3 6 6 4 3 4 5 4 3 6 61 pCAP326 myr-RRHNAHHSTPHPDDR RAD9A 36 3 3 4 6 4 4 6 4 6 4 53 *AS - Activity Score.

TABLE 9 Experiment number 2 3 4 Cell line type MDA-MB-231 SW-480 SKBR3p53^(R280H) p53^(R273H,P309S) p53^(R175H) Group Control TreatmentControl Treatment 325 Control Treatment pCAPs pCAPs pCAPs pCAPs pCAPpCAPs pCAPs Number of 12 18 10 10 10 10 10 samples IVIS average 209%1.1% 275.4% 3.3% 4.4% 1000% 43.7% ratio to day 0 Number of 0 10 0 4 2 00 total regression Samples 1.26 0.27 0.87 0.15 0.12 0.38 0.15 averagesize Samples 1.11 0.29 0.53 0.15 0.10 0.53 0.24 average weight

TABLE 10 SEQ ID Activity NO: Plasmid Sequence Score 17 pCAP8LTFEHYWAQLTS 0 18 pCAP12 GGGGGGGGGGGG 0 19 pCAP19 NPNTYVPHWMRQ 0 20pCAP25 YRRLLIGMMW 0 21 pCAP26 DEFHSFYTARQTG 0 22 pCAP29 KPDSPRV 0 23pCAP31 PPYSQFLQWYLS 0 24 pCAP40 SEFPRSWDMETN 0 25 pCAP45 HDTHNAHVG 0 26pCAP50 WSEYDIPTPQIPP 0 27 pCAP69 SILTLSRRRRRRRRRR 0 28 pCAP73SCRCRLRGDRGDR 0 29 pCAP76 GGGGGGGGGRRRRRRR 0 30 pCAP77 SEYLCSSLDAAG 0 31pCAP78 GESFVQHVFRQN 0 32 pCAP79 SVHHHHRMHLVA 0 33 pCAP84 GRRRFCM 0 34pCAP85 KLTIHHH 0 35 pCAP86 FGSHHEL 0 36 pCAP96 GTVDHHA 0 37 pCAP107DRLSVFLFIM 0 38 pCAP114 AISHHTR 0 39 pCAP116 KHHPFDHRLGNQ 0 40 pCAP119HSAHHTM 0 41 pCAP125 ELGLHRH 0 42 pCAP126 RRLRICV 0 43 pCAP156VPHIHEFTRRRRRRRR 0 44 pCAP164 PLTLI 0 45 pCAP165 SLLIG 0 46 pCAP166KPPER 0 47 pCAP168 CRIIR 0 48 pCAP169 SFILI 0 49 pCAP171 PHHHS 0 50pCAP172 EFHS 0 51 pCAP173 RLRRL 0 52 pCAP175 DSPR 0 53 pCAP176 HPWTH 054 pCAP177 HFSHH 0 55 pCAP178 RRVI 0 56 pCAP179 ILVI 0 57 pCAP207RRSRSNEDVEDKTEDE 0 58 pCAP208 RRIRSGGKDHAWTPLHENH 0 59 pCAP209HTPHPPVARTSPLQTPRR 0 60 pCAP211 PDSEPPRMELRRR 0 61 pCAP215 RRDTFDIRILMAF0 62 pCAP218 RREVTELHHTHEDRR 0 63 pCAP223 SPWTHERRCRQR 0 64 pCAP232RSRSSHLRDHERTHT 0 65 pCAP236 RRRSTNTFLGEDFDQ 0 66 pCAP241 LIGLSTSPRPRIIR0 67 pCAP248 EIYGESGKTDEHALDTEYRR 0 68 pCAP252 RRVILRSYDGGHSTPHPD 0 69pCAP253 TGKTFVKRHLTEFEKKYR 0 70 pCAP254 NHFDYDTIELDTAGEYSRRR 0 71pCAP255 DPEPPRYLPPPPERR 0 72 pCAP260 RRTFIRHRIDSTEVIYQDED 0 73 pCAP262ESKTGHKSEEQRLRRYR 0 74 pCAP263 YDDEHNHHPHHSTHRRR 0 75 pCAP264RRRREVHTIHQHGIVHSD 0 76 pCAP269 DEPLPPPERRR 0 77 pCAP270 SPHPPY 0 78pCAP271 SPHPPYSPHPPYSPHPPYP 0 79 pCAP272 RRPHNLHHD 0 80 pCAP274LRDPHPPERRIR 0 81 pCAP283 RRPADQISYLHPPER 0 82 pCAP291 DLQYDFPRIRR 0 83pCAP292 YDELYQKEDPHRRR 0 84 pCAP294 FKPERFPQNDRRR 0 85 pCAP296 RPADRIRR0 86 pCAP297 HDFDPRYRDRR 0 87 pCAP300 RIRRDPDSPLPHPE 0 88 pCAP304myr-RRIRILMFLIGCGRV 0 89 pCAP309 HPHVILPRIRIRIR 0 90 pCAP311EIHTIHLLPERR 0 91 pCAP320 EPSHPRSRYPRTF 0 92 pCAP321 RNIIIRDFIHFSHIDR 093 pCAP322 RRIRDPQIK-myrLEIHFSHID 0 94 pCAP323 myr-DLHTIHIPRDRR 0 95pCAP324 SHDFPHREPRPERR 0 96 pCAP219 SYRHYSDHWEDRRR 1 97 pCAP2VWVHDSCHANLQNYRNYLLP 2 98 pCAP4 EHDFEVRGDVVNGRNHQGPK 2 99 pCAPS LEVIYMI2 100 pCAP38 WTLSNYL 2 101 pCAP39 DSLHSTY 2 102 pCAP41 WHHRQQIPRPLE 2103 pCAP64 APSIFTPHAWRQ 2 104 pCAP66 THFSHHLKGGGRRQRRRP 2 105 pCAP67LHSKTLVLGGGRRRRGDR 2 106 pCAP71 WTLSNYLGGRKKRRQRRRR 2 107 pCAP81VRCIFRGIWVRL 2 108 pCAP98 HSSGHNFVLVRQ 2 109 pCAP110 LFILVFR 2 110pCAP112 TTSHHPK 2 111 pCAP124 VMVLFRILRGSM 2 112 pCAP162 SILT 2 113pCAP214 RRRESEQRSISLHHHST 2 114 pCAP216 myr-HFNHYTFESTCRRRRC 2 115pCAP217 HSTPHPPQPPERRR 2 116 pCAP224 RRKSEPHSLSGGYQTGAD 2 117 pCAP234HRTGHYTRCRQRCRSRSHNRH 2 118 pCAP243 RRCRSILPLLLLSR 2 119 pCAP256RTLHGRRVILHEGGHSISDLK 2 120 pCAP266 HHRLSYFIVRRHSTHASR 2 121 pCAP293RRIRIDPQHD 2 122 pCAP299 ILQPDFLIRPE 2 123 pCAP307 HDPRIIRIR 2 124pCAP52 SPYPIRT 3 125 pCAP53 ILVIIQRIM 3 126 pCAP101 IRFILIR 3 127pCAP102 SSVHHRG 3 128 pCAP103 LRRQLQL 3 129 pCAP113 HTTAHTH 3 130pCAP115 HPHNHTVHNVVY 3 131 pCAP117 DHSKFVPLFVRQ 3 132 pCAP120SIRTLGRFLIIRV 3 133 pCAP123 GLCRIIL 3 134 pCAP127 SPPIRHH 3 135 pCAP201HPTHPIRLRDNLTR 3 136 pCAP212 myr-REEETILIIRRR 3 137 pCAP225HTIHSISDFPEPPDRRRR 3 138 pCAP228 DEDAAHSTGHPHNSQHRRRR 3 139 pCAP240TEQHHYIPHRRR 3 140 pCAP251 RLRRVILRSYHE 3 141 pCAP265EEPDRQPSGKRGGRKRRSR 3 142 pCAP273 RDFHTIHPSISRR 3 143 pCAP276RRVDIHDGQRR 3 144 pCAP277 DQPYPHRRIR 3 145 pCAP281 myr-RDFILFIRRLGRR 3146 pCAP295 LDLYHPRERR 3 147 pCAP298 RRIRDPLGNEHE 3 148 pCAP303 IVEFRIRR3 149 pCAP312 RRPRIPDYIL 3 150 pCAP314 RSTPHIHEFIRR 3 151 pCAP319SHDFYPHWMRERIR 3 152 pCAP13 HFSHHLK 4 153 pCAP32 TSPLQSLK 4 154 pCAP51AILTLILRRVIWP 4 155 pCAP94 LRFIDYP 4 156 pCAP109 GPIKHHLQHH 4 157pCAP163 LTLS 4 158 pCAP222 RYEENNGVNPPVQVFESRTR 4 159 pCAP239REGFYGPWHEQRRR 4 160 pCAP285 RRDIIRHNAHS 4 161 pCAP286 HDFHDYLERR 4 162pCAP305 IREFDPRRIR 4 163 pCAP310 RLRCLLLLIGRVGRR 4 164 pCAP6LGIDEDEETETAPE 5 165 pCAP22 SLLIGFGIIRSR 5 166 pCAP27 VHEVTHHWL 5 167pCAP56 ATPFHQT 5 168 pCAP58 SILPLFLIRRSG 5 169 pCAP72 SCRCRLRRRRRRRRRR 5170 pCAP105 SRIVLGW 5 171 pCAP111 SNIHHQV 5 172 pCAP121 LTLMRLRIIG 5 173pCAP122 HSYSPYYTFRQH 5 174 pCAP167 FILIR 5 175 pCAP205RCRNRKKEKTECLQKESEK 5 176 pCAP213 RRIKMIRTSESFIQHIVS 5 177 pCAP244RRVSELQRNKHGRKHEL 5 178 pCAP246 RRRLDDEDVQTPTPSEYQN 5 179 pCAP261RRRQPLPSAPENEE 5 180 pCAP7 SPLQTPAAPGAAAGPALSPV 6 181 pCAP18 SHQVHTHHNN6 182 pCAP37 KLQVPIK 6 183 pCAP74 IRGRIIRRKKRRQRRRRGDR 6 184 pCAP82QIPHRSSTALQL 6 185 pCAP88 SYQTMQP 6 186 pCAP140 TDSHSHHRRRRRRRRRRR 6 187pCAP143 IPMNFTSHSLRQRRRRRRRRR 6 188 pCAP153 YWSAPQPATRRRRRRRRRRR 6 189pCAP220 STTHPHPGTSAPEPATRRR 6 190 pCAP226 DDSDNRIIRYRR 6 191 pCAP238TSPHPSLPRHIYPRR 6 192 pCAP247 RRITEIRGRTGKTTLTYIED 6 193 pCAP249myr-DERTGKTRRYIDTRDIRR 6 194 pCAP275 myr-MTYSDMPRRIITDEDRRR 6 195pCAP278 RRYDTVIDDIEYRR 6 196 pCAP279 RDTIERPEIRR 6 197 pCAP280myr-RYRRLILEIWRR 6 198 pCAP284 myr-RHDTHNAHIRR 6 199 pCAP288THDFDRLLRIRRR 6 200 pCAP289 RHNHIRPDNQ 6 201 pCAP290 RYKEPRITPRE 6 202pCAP302 LRIEPIRIR 6 203 pCAP306 myr-RLIRIRILM 6 204 pCAP318RPEFHSFHPIYERR 6 205 pCAP91 STTHIHA 7 206 pCAP92 FPHLVSSLTT 7 207 pCAP99GLHLFTTDRQGW 7 208 pCAP132 NHPWQFPNRWTRRRRRR 7 209 pCAP145HSSHHHPVHSWNRRRRRRR 7 210 pCAP316 myr-DIHTIHLPDTHRR 7 211 pCAP10VAEFAQSIQSRIVEWKERLD 8 212 pCAP49 TRILCIVMM 8 213 pCAP55 FLLPEPDENTRW 8214 pCAP57 LMSNAQY 8 215 pCAP89 SILTLSCRCRLRLWR 8 216 pCAP95 HQIHRNHTY 8217 pCAP106 LIRRCSLQR 8 218 pCAP137 GAMHLPWHMGTRRRRRR 8 219 pCAP202DEDAKFRIRILMRR 8 220 pCAP245 NHITNGGEEDSDCSSRRRRL 8 221 pCAP257myr-HSSHHHPTVQHRR 8 222 pCAP287 RDFERTIVDI 8 223 pCAP313myr-RRREILHPEFRILYE 8 224 pCAP14 HHFSHHWKT 9 225 pCAP59 FLIRRSG 9 226pCAP63 HNHHHSQHTPQH 9 227 pCAP80 HLHKHHYKDSRM 9 228 pCAP231HRTQSTLILFIRRGRET 9 229 pCAP315 LHFSHIDRR 9 230 pCAP62 YELPHHAYPA 10 231pCAP133 SLLIGFGIIRSRRRRRRRR 10 232 pCAP135 HTDSHPHHHHPHRRRRR 10 233pCAP147 ATQHHYIKRRRRRRRRRRR 10 234 pCAP129 FRSFAIPLVVPFRRRRRRR 11 235pCAP138 YPTQGHLRRRRRRRRRRRR 11 236 pCAP146 HANLHHTRRRRRRRRRRR 11 237pCAP152 YRRLLIGMRRRRRRRRRRRR 11 238 pCAP233 SHYHTPQNPPSTRRR 11 239pCAP235 RSYSKLLCLLERLRISP 11 240 pCAP3 FWTQSIKERKMLNEHDFEVR 12 241pCAP15 THFSHHLKH 12 242 pCAP90 SCRCRLR 12 243 pCAP139 MHPPDWYHHTPKRRRRRR12 244 pCAP237 HTIHVHYPGNRQPNPPLILQR 12 245 pCAP268 TPSYGHTPSHHRRR 12246 pCAP301 myr-IRGRIRIIRRIR 12 247 pCAP20 HHPWTHHQRWS 13 248 pCAP48IPMNFTSHSLRQ 13 249 pCAP118 SNHHHRHHTNTH 13 250 pCAP130EVTFRHSVVRRRRRRRRRRR 13 251 pCAP149 FPGHTIHRRRRRRRRRRR 13 252 pCAP34SILTLSRIVLGWW 14 253 pCAP47 TLYLPHWHRH 14 254 pCAP136 SILTLRLRRLRRRRRRRR14 255 pCAP142 TLYLPHWHRHRRRRRRRRRR 14 256 pCAP43 TDSHSHH 15 257 pCAP11EWKERLDKEFSLSVYQKMKF 16 258 pCAP30 TIHPSIS 16 259 pCAP33 SILTLRLRRLRR 16260 pCAP44 VPHIHEFT 16 261 pCAP9 TIIHREDEDEIEW 17 262 pCAP61KDLPFYSHLSRQ 17 263 pCAP65 THFSHHLKHRRRRRRRRRR 17 264 pCAP93 ATQHHYIK 17265 pCAP108 IIRGNFLIGGRL 17 266 pCAP131 LPNPPERHHRRRRRRRRRRR 18 267pCAP158 SFILFIRRGRLGRGDR 18 268 pCAP100 FPGHTIH 19 269 pCAP128 CILRLWW19 270 pCAP206 RRRSHSQENVDQDTDE 19 271 pCAP204 MSTESNMPRLIQNDDRRR 20 272pCAP104 LLRLGLI 21 273 pCAP23 IRILMFLIGCGR 22 274 pCAP17 LHSKTLVL 23 275pCAP24 LRCLLLLIGRVG 23 276 pCAP258 FLIGPDRLIRSR 23 277 pCAP16 LPNPPERHH24 278 pCAP28 HTDSHPHHHHPH 24 279 pCAP160 Fitc-SFILFIRRGRLGRRRRRRRRR 24280 pCAP83 HSSHHHPVHSWN 25 281 pCAP259 myr-RTLIGIIRSHHLTLIRR 25 282pCAP54 IRGRIIR 26 283 pCAP150 IIRGNFLIGGRLRRRRRRRRR 26 284 pCAP170 IRILM27 285 pCAP35 GAMHLPWHMGTL 28 286 pCAP267 KRGGRKRRGGGHRLSYFIRR 28 287pCAP21 NHPWQFPNRWTV 29 288 pCAP42 MHPPDWYHHTPKH 29 289 pCAP141SWQALALYAAGWRRRRRR 29 290 pCAP161 HNAH 29 291 pCAP210 DEFERYRRFSTSRRR 29292 pCAP1 EVTFRHSVV 32 293 pCAP75 TRILCIVRKKRRQRRRRGDR 32 294 pCAP70SILTLSRGRKKRRQRRRR 33 295 pCAP151 CILRLWWRRRRRRRRRRR 33 296 pCAP46ASWQALALYAAGW 34 297 pCAP229 myr-PRVLPSPHTIHPSQYP 36 298 pCAP87 HANLHHT37 299 pCAP157 SFILFIRRGRLGRKKRRQRRRP 37 300 pCAP36 YPTQGHLR 39 301pCAP68 YRRLLIGMMWRRRRRRRRRRR 39 302 pCAP60 SFILFIRRGRLG 40 303 pCAP134IRILMFLIGCGRRRRRRRR 40 304 pCAP308 myr-RRICRFIRICRVR 40 305 pCAP155IRGRIIRRRRRRRRRR 41 306 pCAP203 RRRHDSCHNQLQNYDHSTE 42 307 pCAP148WNHHHSTPHPRRRRRRRRRR 43 308 pCAP282 myr-RRPVAPDLRHTIHIPPER 43 309pCAP317 RRDIHTIHPFYQ 43 310 pCAP97 WNHHHSTPHPAH 47 311 pCAP144SFILFIRRGRLGRRRRRRRRR 49 312 pCAP159 rrrrrrrrglrgrriflifs 53 313 pCAP326myr-RRHNAHHSTPHPDDR 53 314 pCAP174 HS TPHP 54 315 pCAP154LRCLLLLIGRVGRKKRRQRR 57 316 pCAP221 myr-RRKHNKHRPEPDSDER 60 317 pCAP325myr-RRIRDPRILLLHFD 61 318 pCAP230 RKRGKSYAFFVPPSESKERW 63 319 pCAP227myr-RRKILFIRLMHNKH 67 320 pCAP242 myr-RRLIVRILKLPNPPER 67 321 pCAP250myr-RRHSTPHPD 72

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

What is claimed is:
 1. A recombinant or synthetic peptide comprising anamino-acid sequence set forth in any one of SEQ ID NOs:314, 268, 282,340, 376, 298, 377, 378, 253, 20, 379, 302, 275, 380, 273, 381, 280 or382, wherein said peptide at least partially reactivates a mutant p53protein; and wherein said peptide is up to 30 amino-acids in length. 2.The peptide of claim 1, comprising the amino-acid sequence set forth inSEQ ID NO:314.
 3. The peptide of claim 2, comprising the amino-acidsequence set forth in SEQ ID NO:321, SEQ ID NO:314, SEQ ID NO:313, SEQID NO:310 or SEQ ID NO:307.
 4. The peptide of claim 3, consisting of theamino-acid sequence set forth in SEQ ID NO:321, SEQ ID NO:314, SEQ IDNO:313, SEQ ID NO:310 or SEQ ID NO:307.
 5. The peptide of claim 1,conjugated to at least one cell permeation moiety.
 6. The peptide ofclaim 5, wherein said cell permeation moiety is a fatty acid moiety. 7.The peptide of claim 6, wherein said fatty acid moiety is a myristoylfatty acid.
 8. The peptide of claim 5, wherein said cell permeationmoiety is an amino acid moiety.
 9. The peptide of claim 8, wherein saidamino acid moiety is a poly arginine moiety.
 10. The peptide of claim 1,wherein said peptide at least partially changes the conformation of saidmutant p53 protein to a conformation of a wild-type (WT) p53 protein.11. The peptide of claim 1, wherein said peptide at least partiallychanges the conformation of said mutant p53 protein such that saidmutant p53 protein is recognized by a monoclonal antibody directedagainst a WT p53 protein.
 12. The peptide of claim 1, wherein saidmutant p53 protein is not recognized by a monoclonal antibody directedagainst a WT p53 protein.
 13. The peptide of claim 1, wherein saidmutant p53 protein, upon binding to said peptide, is recognized by amonoclonal antibody directed against a WT p53 protein.
 14. The peptideof claim 13, wherein said monoclonal antibody is Ab1620.
 15. The peptideof claim 1, wherein said peptide at least partially restores theactivity of said mutant p53 protein to the activity of a WT p53 protein.16. The peptide of claim 15, wherein said activity is reducing viabilityof cells expressing said mutant p53 protein.
 17. The peptide of claim15, wherein said activity is promoting apoptosis of cells expressingsaid mutant p53 protein.
 18. The peptide of claim 15, wherein saidactivity is binding to a p53 consensus DNA binding element in cellsexpressing said mutant p53 protein.
 19. The peptide of claim 1, whereinsaid mutant p53 protein is of a different conformation than a WT p53protein.
 20. The peptide of claim 1, wherein said mutant p53 protein isat least partly inactive compared to a WT p53 protein.
 21. A recombinantor synthetic peptide comprising the amino-acid sequence set forth in anyone of SEQ ID NOs:321-286, wherein said peptide is up to 50 amino-acidsin length.
 22. The peptide of claim 21, wherein said peptide at leastpartially reactivates a mutant p53 protein.
 23. The peptide of claim 22,comprising the amino-acid sequence set forth in SEQ ID NO:321, SEQ IDNO:314, SEQ ID NO:313, SEQ ID NO:310 or SEQ ID NO:307.
 24. The peptideof claim 21, wherein said peptide is less than 30 amino-acids in length.25. A recombinant or synthetic peptide consisting of the amino-acidsequence set forth in any one of SEQ ID NOs:321-286.
 26. The peptide ofclaim 25, consisting of the amino-acid sequence set forth in NO:321, SEQID NO:314, SEQ ID NO:313, SEQ ID NO:310 or SEQ ID NO:307.
 27. Thepeptide of claim 25, consisting of the amino-acid sequence set forth inany one of SEQ ID NOs:321-302.
 28. The peptide of claim 25, consistingof the amino-acid sequence set forth in any one of SEQ ID NOs:321-312.29. The peptide of claim 25, consisting of the amino-acid sequence setforth in any one of SEQ ID NOs: 321-316.
 30. A method of treating adisease, disorder or condition associated with a mutant p53 protein,comprising the step of administering a therapeutically effective amountof the peptide of claim 1 to a subject in need thereof, thereby treatingsaid disease, disorder or condition.
 31. A recombinant or syntheticpeptide comprising the amino-acid sequence set forth in SEQ ID NO: 321.32. A recombinant or synthetic peptide consisting of the amino-acidsequence set forth in SEQ ID NO: 321.